Compositions and methods for potentiated activity of biologically active molecules

ABSTRACT

The present invention relates to novel compositions and methods for potentiating the activity of biologically active molecules in conjunction with one or more delivery vehicles and one or more carrier molecules. Specifically, the invention features the use of a carrier molecule in combination with a delivery vehicle and a biologically active molecule of interest to potentiate the activity of the biologically active molecule. The carrier molecule can be biologically inert, inactive, or attenuated; or can alternately be biologically active in the same or different manner than the biologically active molecule of interest. Specifically, the invention features novel particle forming delivery agents including cationic lipids, microparticles, and nanoparticles that are useful for delivering various biologically active molecules to cells in conjunction with a carrier molecule. The invention also features compositions, and methods of use for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of gene expression and/or activity in a subject or organism that are delivered intracellularly in conjunction with a carrier molecule. In various embodiments, the invention relates to novel cationic lipids, microparticles, nanoparticles and transfection agents that effectively transfect or deliver biologically active molecules, such as antibodies (e.g., monoclonal, chimeric, humanized etc.), cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, allozymes, aptamers, decoys and analogs thereof, and small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules, to relevant cells and/or tissues, such as in a subject or organism, in conjunction with one or more carrier molecules. Such novel cationic lipids, microparticles, nanoparticles and transfection agents that are used in conjunction with one or more carrier molecules are useful, for example, in providing compositions to prevent, inhibit, or treat diseases, conditions, or traits in a cell, subject or organism.

FIELD OF THE INVENTION

The present invention relates to novel compositions and methods forpotentiating the activity of biologically active molecules inconjunction with one or more delivery vehicles and one or more carriermolecules. Specifically, the invention features the use of a carriermolecule in combination with a delivery vehicle and a biologicallyactive molecule of interest to potentiate the activity of thebiologically active molecule. The carrier molecule can be biologicallyinert, inactive, or attenuated; or can alternately be biologicallyactive in the same or different manner than the biologically activemolecule of interest. Specifically, the invention features novelparticle forming delivery agents including cationic lipids,microparticles, and nanoparticles that are useful for delivering variousbiologically active molecules to cells in conjunction with a carriermolecule. The invention also features compositions, and methods of usefor the study, diagnosis, and treatment of traits, diseases andconditions that respond to the modulation of gene expression and/oractivity in a subject or organism that are delivered intracellularly inconjunction with a carrier molecule. In various embodiments, theinvention relates to novel cationic lipids, microparticles,nanoparticles and transfection agents that effectively transfect ordeliver biologically active molecules, such as antibodies (e.g.,monoclonal, chimeric, humanized etc.), cholesterol, hormones,antivirals, peptides, proteins, chemotherapeutics, small molecules,vitamins, co-factors, nucleosides, nucleotides, oligonucleotides,enzymatic nucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, allozymes, aptamers, decoys andanalogs thereof, and small nucleic acid molecules, such as shortinterfering nucleic acid (siNA), short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA(shRNA) molecules, to relevant cells and/or tissues, such as in asubject or organism, in conjunction with one or more carrier molecules.Such novel cationic lipids, microparticles, nanoparticles andtransfection agents that are used in conjunction with one or morecarrier molecules are useful, for example, in providing compositions toprevent, inhibit, or treat diseases, conditions, or traits in a cell,subject or organism.

BACKGROUND OF THE INVENTION

The present invention relates to novel compositions and methods forpotentiating the activity of biologically active molecules in vitro andin vivo. Specifically, the invention relates to compounds, compositionsand methods for delivering nucleic acids, polynucleotides, andoligonucleotides such RNA, DNA and analogs thereof, peptides,polypeptides, proteins, antibodies, hormones and small molecules, tocells by facilitating transport across cellular membranes in, forexample, epithelial tissues and endothelial tissues by using one or moredelivery vehicles and one or more carrier molecules. The compounds,compositions and methods of the invention are useful in therapeutic,research, and diagnostic applications that rely upon the efficienttransfer of biologically active molecules into cells, tissues, andorgans. The discussion is provided only for understanding of theinvention that follows. This summary is not an admission that any of thework described below is prior art to the claimed invention.

The cellular delivery of various therapeutic compounds, such asantiviral and chemotherapeutic agents, is usually compromised by twolimitations. First the selectivity of a number of therapeutic agents isoften low, resulting in high toxicity to normal tissues. Secondly, thetrafficking of many compounds into living cells is highly restricted bythe complex membrane systems of the cell. Specific transporters allowthe selective entry of nutrients or regulatory molecules, whileexcluding most exogenous molecules such as nucleic acids and proteins.Various strategies can be used to improve transport of compounds intocells, including the use of lipid carriers, biodegradable polymers, andvarious conjugate systems.

The most well studied approaches for improving the transport of foreignnucleic acids into cells involve the use of viral vectors or cationiclipids and related cytofectins. Viral vectors can be used to transfergenes efficiently into some cell types, but they generally cannot beused to introduce chemically synthesized molecules into cells. Analternative approach is to use delivery formulations incorporatingcationic lipids, which interact with nucleic acids through one end andlipids or membrane systems through another (for a review see Felgner,1990, Advanced Drug Delivery Reviews, 5, 162-187; Felgner 1993, J.Liposome Res., 3, 3-16). Synthetic nucleic acids as well as plasmids canbe delivered using the cytofectins, although the utility of suchcompounds is often limited by cell-type specificity, requirement for lowserum during transfection, and toxicity.

Another approach to delivering biologically active molecules involvesthe use of conjugates. Conjugates are often selected based on theability of certain molecules to be selectively transported into specificcells, for example via receptor-mediated endocytosis. By attaching acompound of interest to molecules that are actively transported acrossthe cellular membranes, the effective transfer of that compound intocells or specific cellular organelles can be realized. Alternately,molecules that are able to penetrate cellular membranes without activetransport mechanisms, for example, various lipophilic molecules, can beused to deliver compounds of interest. Examples of molecules that can beutilized as conjugates include but are not limited to peptides,hormones, fatty acids, vitamins, flavonoids, sugars, reporter molecules,reporter enzymes, chelators, porphyrins, intercalcators, and othermolecules that are capable of penetrating cellular membranes, either byactive transport or passive transport.

The delivery of compounds to specific cell types, for example, cancercells or cells specific to particular tissues and organs, can beaccomplished by utilizing receptors associated with specific cell types.Particular receptors are overexpressed in certain cancerous cells,including the high affinity folic acid receptor. For example, the highaffinity folate receptor is a tumor marker that is overexpressed in avariety of neoplastic tissues, including breast, ovarian, cervical,colorectal, renal, and nasoparyngeal tumors, but is expressed to a verylimited extent in normal tissues. The use of folic acid based conjugatesto transport exogenous compounds across cell membranes can provide atargeted delivery approach to the treatment and diagnosis of disease andcan provide a reduction in the required dose of therapeutic compounds.Furthermore, therapeutic bioavailability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use ofbioconjugates, including folate bioconjugates. Godwin et al., 1972, J.Biol. Chem., 247, 2266-2271, report the synthesis of biologically activepteroyloligo-L-glutamates. Habus et al., 1998, Bioconjugate Chem., 9,283-291, describe a method for the solid phase synthesis of certainoligonucleotide-folate conjugates. Cook, U.S. Pat. No. 6,721,208,describes certain oligonucleotides modified with specific conjugategroups. The use of biotin and folate conjugates to enhance transmembranetransport of exogenous molecules, including specific oligonucleotideshas been reported by Low et al., U.S. Pat. Nos. 5,416,016, 5,108,921,and International PCT publication No. WO 90/12096. Manoharan et al.,International PCT publication No. WO 99/66063 describe certain folateconjugates, including specific nucleic acid folate conjugates with aphosphoramidite moiety attached to the nucleic acid component of theconjugate, and methods for the synthesis of these folate conjugates.Nomura et al., 2000, J. Org. Chem., 65, 5016-5021, describe thesynthesis of an intermediate,alpha-[2-(trimethylsilyl)ethoxycarbonyl]folic acid, useful in thesynthesis of ceratin types of folate-nucleoside conjugates. Guzaev etal., U.S. Pat. No. 6,335,434, describes the synthesis of certain folateoligonucleotide conjugates. Vargeese et al., International PCTPublication No. WO 02/094185 and U.S. Patent Application PublicationNos. 20030130186 and 20040110296 describe certain nucleic acidconjugates.

The delivery of compounds to other cell types can be accomplished byutilizing receptors associated with a certain type of cell, such ashepatocytes. For example, drug delivery systems utilizingreceptor-mediated endocytosis have been employed to achieve drugtargeting as well as drug-uptake enhancement. The asialoglycoproteinreceptor (ASGPr) (see for example Wu and Wu, 1987, J. Biol. Chem. 262,4429-4432) is unique to hepatocytes and binds branchedgalactose-terminal glycoproteins, such as asialoorosomucoid (ASOR).Binding of such glycoproteins or synthetic glycoconjugates to thereceptor takes place with an affinity that strongly depends on thedegree of branching of the oligosaccharide chain, for example,triatennary structures are bound with greater affinity than biatenarryor monoatennary chains (Baenziger and Fiete, 1980, Cell, 22, 611-620;Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee and Lee, 1987,Glycoconjugate J., 4, 317-328, obtained this high specificity throughthe use of N-acetyl-D-galactosamine as the carbohydrate moiety, whichhas higher affinity for the receptor, compared to galactose. This“clustering effect” has also been described for the binding and uptakeof mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom etal., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose andgalactosamine based conjugates to transport exogenous compounds acrosscell membranes can provide a targeted delivery approach to the treatmentof liver disease such as HBV and HCV infection or hepatocellularcarcinoma. The use of bioconjugates can also provide a reduction in therequired dose of therapeutic compounds required for treatment.Furthermore, therapeutic bioavailability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use ofbioconjugates.

A number of peptide based cellular transporters have been developed byseveral research groups. These peptides are capable of crossing cellularmembranes in vitro and in vivo with high efficiency. Examples of suchfusogenic peptides include a 16-amino acid fragment of the homeodomainof ANTENNAPEDIA, a Drosophila transcription factor (Wang et al., 1995,PNAS USA., 92, 3318-3322); a 17-mer fragment representing thehydrophobic region of the signal sequence of Kaposi fibroblast growthfactor with or without NLS domain (Antopolsky et al., 1999, Bioconj.Chem., 10, 598-606); a 17-mer signal peptide sequence of caimancrocodylus Ig(5) light chain (Chaloin et al., 1997, Biochem. Biophys.Res. Comm., 243, 601-608); a 17-amino acid fusion sequence of HIVenvelope glycoprotein gp4114, (Morris et al., 1997, Nucleic Acids Res.,25, 2730-2736); the HIV-1 Tat49-57 fragment (Schwarze et al., 1999,Science, 285, 1569-1572); a transportan A-achimeric 27-mer consisting ofN-terminal fragment of neuropeptide galanine and membrane interactingwasp venom peptide mastoporan (Lindgren et al., 2000, BioconjugateChem., 11, 619-626); and a 24-mer derived from influenza virushemagglutinin envelop glycoprotein (Bongartz et al., 1994, Nucleic AcidsRes., 22, 4681-4688). These peptides were successfully used as part ofan antisense oligodeoxyribonucleotide-peptide conjugate for cell culturetransfection without lipids. In a number of cases, such conjugatesdemonstrated better cell culture efficacy then parent oligonucleotidestransfected using lipid delivery. In addition, use of phage displaytechniques has identified several organ targeting and tumor targetingpeptides in vivo (Ruoslahti, 1996, Ann. Rev. Cell Dev. Biol., 12,697-715). Conjugation of tumor targeting peptides to doxorubicin hasbeen shown to significantly improve the toxicity profile and hasdemonstrated enhanced efficacy of doxorubicin in the in vivo murinecancer model MDA-MB-435 breast carcinoma (Arap et al., 1998, Science,279, 377-380).

Another approach to the intracellular delivery of biologically activemolecules involves the use of cationic polymers. For example, Ryser etal., International PCT Publication No. WO 79/00515 describes the use ofhigh molecular weight lysine polymers for increasing the transport ofvarious molecules across cellular membranes. Rothbard et al.,International PCT Publication No. WO 98/52614, describes certain methodsand compositions for transporting drugs and macromolecules acrossbiological membranes in which the drug or macromolecule is covalentlyattached to a transport polymer consisting of from 6 to 25 subunits, atleast 50% of which contain a guanidino or amidino side chain. Thetransport polymers are preferably polyarginine peptides composed of allD-, all L- or mixtures of D- and L-arginine. Rothbard et al., U.S.Patent Application Publication No. 20030082356, describescertain-poly-lysine and poly-arginine compounds for the delivery ofdrugs and other agents across epithelial tissues, including the skin,gastrointestinal tract, pulmonary epithelium and blood brain barrier.Wendel et al., U.S. Patent Application Publication No. 20030032593,describes certain polyarginine compounds. Rothbard et al., U.S. PatentApplication Publication No. 20030022831, describes certain poly-lysineand poly-arginine compounds for intra-ocular delivery of drugs. Kosak,U.S. Patent Application Publication No. 20010034333, describes certaincyclodextran polymers compositions that include a cross-linked cationicpolymer component. Beigelman et al., U.S. Pat. No. 6,395,713; Reynoldset al., International PCT Publication No. WO 99/04819; Beigelman et al.,International PCT Publication No. WO 99/05094; and Beigelman et al.,U.S. Patent Application Publication No. 20030073640 describe certainlipid based formulations.

Another approach to the intracellular delivery of biologically activemolecules involves the use of liposomes or other particle formingcompositions. Since the first description of liposomes in 1965, byBangham (J. Mol. Biol. 13, 238-252), there has been a sustained interestand effort in the area of developing lipid-based carrier systems for thedelivery of pharmaceutically active compounds. Liposomes are attractivedrug carriers since they protect biological molecules from degradationwhile improving their cellular uptake. One of the most commonly usedclasses of liposome formulations for delivering polyanions (e.g., DNA)is that which contains cationic lipids. Lipid aggregates can be formedwith macromolecules using cationic lipids alone or including otherlipids and amphiphiles such as phosphatidylethanolamine. It is wellknown in the art that both the composition of the lipid formulation aswell as its method of preparation have effect on the structure and sizeof the resultant anionic macromolecule-cationic lipid aggregate. Thesefactors can be modulated to optimize delivery of polyanions to specificcell types in vitro and in vivo. The use of cationic lipids for cellulardelivery of biologically active molecules has several advantages. Theencapsulation of anionic compounds using cationic lipids is essentiallyquantitative due to electrostatic interaction. In addition, it isbelieved that the cationic lipids interact with the negatively chargedcell membranes initiating cellular membrane transport (Akhtar et al.,1992, Trends Cell Bio., 2, 139; Xu et al., 1996, Biochemistry 35, 5616).

Experiments have shown that plasmid DNA can be encapsulated in smallparticles that consist of a single plasmid encapsulated within a bilayerlipid vesicle (Wheeler, et al., 1999, Gene Therapy 6, 271-281). Theseparticles typically contain the fusogenic lipiddioleoylphosphatidylethanolamine (DOPE), low levels of a cationic lipid,and can be stabilized in aqueous media by the presence of apoly(ethylene glycol) (PEG) coating. These particles have systemicapplications as they exhibit extended circulation lifetimes followingintravenous (i.v.) injection, can accumulate preferentially in varioustissues and organs or tumors due to the enhanced vascular permeabilityin such regions, and can be designed to escape the lyosomic pathway ofendocytosis by disruption of endosomal membranes. These properties canbe useful in delivering biologically active molecules to various celltypes for experimental and therapeutic applications. For example, theeffective use of nucleic acid technologies such as short interfering RNA(siRNA), antisense, ribozymes, decoys, triplex forming oligonucleotides,2-5A oligonucleotides, and aptamers in vitro and in vivo may benefitfrom efficient delivery of these compounds across cellular membranes.Lewis et al., U.S. Patent Application Publication No. 20030125281,describes certain compositions consisting of the combination of siRNA,certain amphipathic compounds, and certain polycations. MacLachlan, U.S.Patent Application Publication No. 20030077829, describes certain lipidbased formulations. MacLachlan, International PCT Publication No. WO05/007196, describes certain lipid encapsulated interfering RNAformulations. Vargeese et al., International PCT Publication No.WO2005007854 describes certain polycationic compositions for thecellular delivery of polynucleotides. McSwiggen et al., InternationalPCT Publication Nos. WO 05/019453, WO 03/70918, WO 03/74654 and U.S.Patent Application Publication Nos. 20050020525 and 20050032733,describes short interfering nucleic acid molecules (siNA) and varioustechnologies for the delivery of siNA molecules and otherpolynucleotides.

In addition, recent work involving cationic lipid particles demonstratedthe formation of two structurally different complexes comprising nucleicacid (or other polyanionic compound) and cationic lipid (Safinya et al.,Science, 281: 78-81 (1998). One structure comprises a multilamellarstructure with nucleic acid monolayers sandwiched between cationic lipidbilayers (“lamellar structure”) (FIG. 13). A second structure comprisesa two dimensional hexagonal columnar phase structure (“invertedhexagonal structure”) in which nucleic acid molecules are encircled bycationic lipid in the formation of a hexagonal structure (FIG. 13).Safinya et al. demonstrated that the inverted hexagonal structuretransfects mammalian cells more efficiently than the lamellar structure.Further, optical microscopy studies showed that the complexes comprisingthe lamellar structure bind stably to anionic vesicles without fusing tothe vesicles, whereas the complexes comprising the inverted hexagonalstructure are unstable and rapidly fuse to the anionic vesicles,releasing the nucleic acid upon fusion.

The structural transformation from lamellar phase to inverted hexagonalphase complexes is achieved either by incorporating a suitable helperlipid that assists in the adoption of an inverted hexagonal structure orby using a co-surfactant, such as hexanol. However, neither of thesetransformation conditions are suitable for delivery in biologicalsystems. Furthermore, while the inverted hexagonal complex exhibitsgreater transfection efficiency, it has very poor serum stabilitycompared to the lamellar complex. Thus, there remains a need to designdelivery agents that are serum stable, i.e. stable in circulation, thatcan undergo structural transformation, for example from lamellar phaseto inverse hexagonal phase, under biological conditions.

The present application provides compounds, compositions and methods forsignificantly improving the efficiency of systemic and local delivery ofbiologically active molecules in conjunction with one or more carriermolecules. Among other things, the present application providescompounds, compositions and methods for making and using novel deliveryagents that are stable in circulation and undergo structural changesunder appropriate physiological conditions (e.g., pH) which increase theefficiency of delivery of biologically active molecules in conjunctionwith one or more carrier molecules.

Various lipid nucleic acid particles and methods of preparation thereofare described in U.S. Patent Application Publication Nos. 20030077829,20030108886, 20060051405, 20060083780, 20030104044, 20060051405,20040142025, 200600837880, 20050064595, 2005/0175682, 2005/0118253,20050255153 and 20050008689; and U.S. Pat. Nos. 5,885,613; 6,586,001;6,858,225; 6,858,224; 6,815,432; 6,586,410; 6,534,484; and 6,287,591.

Vagle et al., U.S. Patent Application Publication No. 20060240554describes lipid nanoparticle based compositions and methods for thedelivery of biologically active molecules.

Balmain et al., 1982, Nucleic Acids Research, 10(14): 4259-4277,describes a general method for isolating double-stranded cDNA by ethanolprecipitation following the addition of yeast tRNA carrier.

Strain et al., 1985, Biochem J., 225(2): 529-533, describes theenhancement of DNA-mediated gene transfer by high-Mr carrier DNA insynchronized CV-1 cells.

SUMMARY OF THE INVENTION

The present invention relates to novel compositions and methods forpotentiating the activity of biologically active molecules.Specifically, the invention features compositions comprising deliveryvehicles that include one or more carrier molecules and/or one or morebiologically active molecules. The compositions of the inventionpotentiate the activity and/or intracellular delivery of thebiologically active molecule(s), thereby providing for equivalentbiologic activity with substantially reduced concentrations or doses ofthe biologically active molecule(s). The carrier molecule can bebiologically inert, inactive, or attenuated; or can alternately bebiologically active in the same or different manner than thebiologically active molecule of interest. The novel compositions andmethods for potentiating the intracellular delivery of biologicallyactive molecules can be utilized in both in vitro and in vivoapplications.

In one embodiment, the invention features a composition comprising afirst vehicle including one or more biologically active molecules, and asecond vehicle including one or more carrier molecules, for example as aheterogeneous population. In another embodiment, the first vehicle andthe second vehicle are the same with the exception of the biologicallyactive molecule(s) and the carrier molecule(s) (designated FormulationType A1, see FIG. 1A). In yet another embodiment, the first vehicle andthe second vehicle are different (designated Formulation Type A2, seeFIG. 1B). In one embodiment, the first vehicle comprises at least two(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) different biologically activemolecules.

In one embodiment, the invention features a composition comprising avehicle including one or more biologically active molecules and one ormore carrier molecules, for example as a homogeneous population(designated Formulation Type B, see FIG. 2). In one embodiment, thecomposition comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore) different biologically active molecules.

In one embodiment, the invention features a composition comprising oneor more carrier molecules, and a vehicle including one or morebiologically active molecules, for example as a heterogeneous population(designated Formulation Type C, see FIG. 3). In one embodiment, thecomposition comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore) different biologically active molecules.

In one embodiment, the invention features a composition comprising afirst formulation including one or more carrier molecules and a secondformulation including one or more biologically active molecules (e.g., apolynucleotide such as a siNA, miRNA, RNAi inhibitor, antisense,aptamer, decoy, ribozyme, 2-5A, triplex forming oligonucleotide, othernucleic acid molecule and/or other biologically active moleculedescribed herein), a cationic lipid, a neutral lipid, and apolyethyleneglycol conjugate, such as a PEG-diacylglycerol,PEG-diacylglycamide, PEG-cholesterol, or PEG-DMB conjugate. In anotherembodiment, the first and/or second formulation further comprisescholesterol or a cholesterol derivative. In another embodiment, thefirst and/or second formulation further comprises an alcohol orsurfactant. In another embodiment, the first and/or second formulationfurther comprises lineoyl alcohol. This composition is generallyreferred to herein as LNP Formulation Type A (see FIG. 4). In oneembodiment, the second formulation comprises at least two (e.g., 2, 3,4, 5, 6, 7, 8, 9, 10 or more) different biologically active molecules.

In one embodiment, the invention features a composition comprising aformulation including one or more carrier molecules, one or morebiologically active molecules (e.g., a polynucleotide such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, other nucleic acid molecule and/orother biologically active molecule described herein), a cationic lipid,a neutral lipid, and a polyethyleneglycol conjugate, such as aPEG-diacylglycerol, PEG-diacylglycamide, PEG-cholesterol, or PEG-DMBconjugate. In another embodiment, the formulation further comprisescholesterol or a cholesterol derivative. In another embodiment, theformulation further comprises an alcohol or surfactant. In anotherembodiment, the formulation further comprises lineoyl alcohol. Thiscomposition is generally referred to herein as LNP Formulation Type B(see FIG. 5). In one embodiment, the composition comprises at least two(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) different biologically activemolecules.

In one embodiment, the invention features a composition comprising oneor more carrier molecules, and a formulation including one or morebiologically active molecules (e.g., a polynucleotide such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, other nucleic acid molecule and/orother biologically active molecule described herein), a cationic lipid,a neutral lipid, and a polyethyleneglycol conjugate, such as aPEG-diacylglycerol, PEG-diacylglycamide, PEG-cholesterol, or PEG-DMBconjugate. In another embodiment, the formulation further comprisescholesterol or a cholesterol derivative. In another embodiment, theformulation further comprises an alcohol or surfactant. In anotherembodiment, the formulation further comprises lineoyl alcohol. Thiscomposition is generally referred to herein as LNP Formulation Type C(see FIG. 6). In one embodiment, the composition comprises at least two(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) different biologically activemolecules.

In one embodiment, a biologically active molecule of the invention iscomprises one or more nucleosides, nucleotides, oligonucleotides,enzymatic nucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, allozymes, aptamers, decoys, or smallnucleic acid molecules, including short interfering nucleic acid (siNA),short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA(miRNA), short hairpin RNA (shRNA), RNAi inhibitor molecules and/or anycombination thereof (see for example PCT/US06/032168, incorporated byreference herein in its entirety).

In one embodiment, a biologically active molecule of the inventioncomprises one or more antibodies (including monoclonal, chimeric,humanized etc.), hormones, antivirals, peptides, proteins, vaccines,antibiotics, chemotherapeutics, small molecules, vitamins, and/orco-factors.

In one embodiment, a carrier molecule of the invention comprises one ormore lipids (e.g., cationic lipids, neutral lipids), peptides, proteins,steroids (e.g., cholesterol, estrogen, testosterone, progesterone,glucocortisone, adrenaline, insulin, glucagon, cortisol, vitamin D,thyroid hormone, retinoic acid, and/or growth hormones), smallmolecules, vitamins, co-factors, nucleosides, nucleotides,polynucleotides (e.g., single, double, or triple stranded), and/orpolymers as are generally recognized in the art, or any combinationthereof. In one embodiment, a polynucleotide based carrier molecule ofthe invention comprises one or more nucleic acid molecules, includingsingle stranded RNA or DNA molecules, for example from about 2 to about100,000 bases in length; double stranded RNA or DNA molecules, forexample from about 2 to about 100,000 base pairs in length, or triplexRNA or DNA molecules, for example from about 2 to about 100,000 basepairs in length. In one embodiment, a polynucleotide based carriermolecule of the invention comprises a non-human DNA derived from adivergent species, such as non-human sperm DNA (see for exampleJP63102682, describing salmon sperm DNA). In another embodiment, apolynucleotide based carrier molecule of the invention comprises anon-human RNA derived from a divergent species, such as non-human tRNA.In one embodiment, a polynucleotide carrier molecule is a shortinterfering nucleic acid (siNA) molecule as described herein. In anotherembodiment, a polynucleotide carrier molecule is not complementary to atarget nucleic acid molecule which is targeted by a biologically activemolecule within the same composition. For example, if a biologicallyactive molecule of the invention comprises a siNA molecule that hascomplementarity to a target polynucleotide sequence, then a nucleic acidbased carrier molecule utilized in a composition of the invention wouldcomprise sequence that does not have complementarity to the targetpolynucleotide sequence. In one embodiment, the carrier molecule of theinvention is a component of a formulation of the invention.

In one embodiment, a double stranded carrier molecule of the inventionis designed so that it is not a good substrate for RISC loading. Forexample, the double stranded carrier molecule can be chemically modifiedso as not to be a substrate for RISC, such as through incorporation ofone or more terminal cap moieties (e.g., on the 5′-end, 3′-end or both5′ and 3′-ends of one or both strands of the double stranded carriermolecule), or through chemical modification of one or more nucleotidesin the double stranded carrier molecule (e.g., incorporation of2′-substituted nucleotides including 2′-O-alkyl, 2′-deoxy,2′-deoxy-2′-fluoro or any other modification herein). In anotherembodiment, the double stranded carrier molecule is designed do that itssequence is not amenable to RISC loading, such as by increasing the Tmof one or more base pairs at one or both ends of the double strandedcarrier molecule.

In one embodiment, a vehicle of the invention is a compositioncomprising one or more transfection agents, liposomes, microparticles,nanoparticles, capsids, viroids, virions, virus like particles (VLP),protein cages, ferritins, hydrogels, or polymers as described herein oras are generally recognized in the art.

In one embodiment, a vehicle of the invention comprises one or morelipid nanoparticle or LNP compositions, see for example LNP compositionsdescribed herein (see for example Table IV) and in U.S. PatentApplication Publication No. 20060240554 and U.S. Ser. No. 11/586,102,filed Oct. 24, 2006, both of which are incorporated by reference hereinin their entirety.

In one embodiment, a vehicle of the invention comprises one or morestable nucleic acid particle or SNALP compositions, see for exampleInternational PCT Publication No. WO2007012191, and U.S. PatentApplication Publication Nos. 2006083780, 2006051405, US2005175682,US2004142025, US2003077829, US2006240093, all of which are incorporatedby reference herein in their entirety.

In one embodiment, a vehicle of the invention comprises one or moredelivery systems as described in International PCT Publication Nos.WO2005105152 and WO2007014391, and U.S. Pat. Nos. 7,148,205, 7,144,869,7,138,382, 7,101,995, 7,098,032, 7,098,030, 7,094,605, 7,091,041,7,087,770, 7,071,163, 7,049,144, 7,049,142, 7,045,356, 7,033,607,7,022,525, 7,019,113, 7,015,040, 6,936,729, 6,919,091, 6,897,068,6,881,576, 6,872,519, 6,867,196, 6,818,626, 6,794,189, 6,740,643,6,740,336, 6,706,922, 6,673,612, 6,630,351, 6,627,616, 6,593,465,6,458,382, 6,429,200, 6,383,811, 6,379,966, 6,339,067, 6,265,387,6,262,252, 6,180,784, 6,126,964, 6,093,701, and 5,744,335; all of whichare incorporated by reference herein in their entirety.

In one embodiment, a vehicle of the invention comprises one or morepeptide or peptide related delivery systems, see for example U.S. PatentApplication Publication Nos. 20060040882, 20050136437, 20050031549, and20060062758, all of which are incorporated by reference herein in theirentirety.

In one embodiment, a vehicle of the invention comprises proteins such asalbumin, collagen, and gelatin, polysaccharides such as dextrans andstarches, and matrix forming compositions including polylactide (PLA),polyglycolide (PGA), lactide-glycolide copolymers (PLG),poly(lactic-co-glycolic acid) (PLGA), polycaprolactone,lactide-caprolactone copolymers, polyhydroxybutyrate,polyalkylcyanoacrylates, polyanhydrides, polyorthoesters, acrylatepolymers and copolymers such as methyl methacrylate, methacrylic acid,hydroxyalkyl acrylates and methacrylates, ethylene glycoldimethacrylate, acrylamide and/or bisacrylamide, cellulose-basedpolymers, ethylene glycol polymers and copolymers, oxyethylene andoxypropylene polymers, poly(vinyl alcohol), polyvinylacetate,polyvinylpyrrolidone, polyvinylpyridine, and/or any combination thereof.

In various embodiments, the invention relates to novel cationic lipids,microparticles, nanoparticles and transfection agents that effectivelytransfect or deliver biologically active molecules, such as antibodies(e.g., monoclonal, chimeric, humanized etc.), cholesterol, hormones,antivirals, peptides, proteins, chemotherapeutics, small molecules,vitamins, co-factors, nucleosides, nucleotides, oligonucleotides,enzymatic nucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, allozymes, aptamers, decoys andanalogs thereof, and small nucleic acid molecules, such as shortinterfering nucleic acid (siNA), short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA(shRNA) molecules, to relevant cells and/or tissues, such as in asubject or organism, in conjunction with one or more carrier molecules.Such novel cationic lipids, microparticles, nanoparticles andtransfection agents that are used in conjunction with one or morecarrier molecules are useful, for example, in providing compositions toprevent, inhibit, or treat diseases, conditions, or traits in a cell,subject or organism.

In one embodiment, the present invention features carrier compounds,compositions, and methods to facilitate delivery of various biologicallyactive molecules into a biological system, such as cells. The carriercompounds, compositions, and methods provided by the instant inventioncan impart therapeutic activity by potentiating the transfer oftherapeutic compounds across cellular membranes or across one or morelayers of epithelial or endothelial tissue. The use of such carriercompounds, compositions, and methods will allow for potentiatedintracellular delivery of biologically active molecules, thus enablingthe use of substantially lower doses of active compounds or alternatelyenabling higher doses of active compounds with fewer side effects.

In one embodiment, the present invention encompasses the design andsynthesis of novel agents for the delivery of biologically activemolecules, including but not limited to small molecules, lipids,nucleosides, nucleotides, nucleic acids, polynucleotides,oligonucleotides, antibodies, toxins, negatively charged polymers andother polymers, for example proteins, peptides, hormones, carbohydrates,or polyamines, across cellular membranes in conjunction with one or morecarrier compounds or compositions. Non-limiting examples ofpolynucleotides that can be delivered across cellular membranes usingthe compounds and methods of the invention include short interferingnucleic acids (siNA) (which includes siRNAs), antisenseoligonucleotides, enzymatic nucleic acid molecules,2′,5′-oligoadenylates, triplex forming oligonucleotides, aptamers,decoys, and cDNA for gene therapy applications. In general, thetransporters described are designed to be used either individually or aspart of a multi-component system, with or without degradable linkers.The compounds of the invention, when formulated into compositions, areexpected to improve delivery of molecules into a number of cell typesoriginating from different tissues, in the presence or absence of serum.

The compounds, compositions, and methods of the invention are useful fordelivering biologically active molecules (e.g., siNAs, siRNAs, miRNAs,siRNA and miRNA inhibitors, nucleic acids, polynucleotides,oligonucleotides, peptides, polypeptides, proteins, hormones,antibodies, and small molecules) to cells or across epithelial andendothelial tissues, such as skin, mucous membranes, vasculaturetissues, gastrointestinal tissues, blood brain barrier tissues,opthalmological tissues, pulmonary tissues, liver tissues, cardiactissues, kidney tissues etc. The compounds, compositions, and methods ofthe invention can be used both for delivery to a particular site ofadministration or for systemic delivery.

The compounds, compositions, and methods of the invention can increasedelivery or availability of biologically active molecules (e.g., siNAs,siRNAs, miRNAs, siRNA and miRNA inhibitors, nucleic acids,polynucleotides, oligonucleotides, peptides, polypeptides, proteins,hormones, antibodies, and small molecules) to cells or tissues comparedto delivery of the molecules in the absence of the compounds,compositions, and methods of the invention. As such, the level of abiologically active molecule inside a cell, tissue, or organism isincreased in the presence of the compounds and compositions of theinvention compared to when the compounds and compositions of theinvention are absent.

In one aspect, the invention features novel cationic lipids,transfection agents, microparticles, nanoparticles, and formulationsthereof with biologically active molecules in conjunction with one ormore carrier molecules. In another embodiment, the invention featurescompositions, and methods of use for the study, diagnosis, and treatmentof traits, diseases, and conditions that respond to the modulation ofgene expression and/or activity in a subject or organism. In anotherembodiment, the invention features novel cationic lipids,microparticles, nanoparticles transfection agents, and formulations thateffectively transfect or deliver small nucleic acid molecules, such asshort interfering nucleic acid (siNA), short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA(shRNA) molecules, and inhibitors thereof (RNAi inhibitors); to relevantcells and/or tissues, such as in a subject or organism in conjunctionwith one or more carrier molecules. Such novel formulations comprisingcarrier compositions, cationic lipids, microparticles, nanoparticles,transfection agents, and formulations are useful, for example, inproviding compositions to prevent, inhibit, or treat diseases,conditions, or traits in a cell, subject or organism as describedherein.

In one aspect, the instant invention features various cationic lipids,microparticles, nanoparticles, transfection agents, and formulations forthe delivery of chemically-modified synthetic short interfering nucleicacid (siNA) molecules and/or RNAi inhibitors that modulate target geneexpression or activity in cells, tissues, such as in a subject ororganism, by RNA interference (RNAi) in conjunction with one or morecarrier molecules. The use of chemically-modified siNA improves variousproperties of native siRNA molecules through increased resistance tonuclease degradation in vivo, improved cellular uptake, and improvedpharmacokinetic properties in vivo. The use of carrier molecules canimprove cellular uptake, fusogenicity, and/or endosomal release of thetherapeutic payload (e.g., siNA), thus enabling a lower dose of activetherapeutic compositions for the same therapeutic effect in vitro and/orin vivo. The carrier molecules, cationic lipids, microparticles,nanoparticles, transfection agents, formulations, and siNA molecules andRNAi inhibitors of the instant invention provide useful reagents andmethods for a variety of therapeutic, veterinary, diagnostic, targetvalidation, genomic discovery, genetic engineering, and pharmacogenomicapplications.

In one aspect, the invention features compositions and methods thatindependently or in combination modulate the expression of target genesencoding proteins, such as proteins associated with the maintenanceand/or development of a disease, trait, or condition, such as a liverdisease, trait, or condition. These genes are referred to hereingenerally as target genes. Such target genes are generally known in theart and transcripts of such genes are commonly referenced by GenbankAccession Number, see for example International PCT Publication No. WO03/74654, serial No. PCT/US03/05028, and U.S. patent application Ser.No. 10/923,536 both incorporated by reference herein). The descriptionbelow of the various aspects and embodiments of the invention isprovided with reference to exemplary target genes and target genetranscripts. However, the various aspects and embodiments are alsodirected to other target genes, such as gene homologs, gene transcriptvariants, and gene polymorphisms (e.g., single nucleotide polymorphism,(SNPs)) that are associated with certain target genes. As such, thevarious aspects and embodiments are also directed to other genes thatare involved in pathways of signal transduction or gene expression thatare involved, for example, in the maintenance and/or development of adisease, trait, or condition. These additional genes can be analyzed fortarget sites using the methods described for target genes herein. Thus,the modulation of other genes and the effects of such modulation of theother genes can be performed, determined, and measured as describedherein.

In one embodiment, the invention features a composition comprising afirst lipid nanoparticle (LNP) vehicle and a second lipid nanoparticle(LNP) vehicle each having size between about 10 nm and 1000 nm, wherein:the first vehicle further comprises one or more biologically activemolecules; the second vehicle further comprises one or more carriermolecules; and each vehicle comprises a cationic lipid, a neutral lipid,and a PEG-lipid. In one embodiment, each lipid nanoparticle vehiclecomprises the same composition of lipid components. In anotherembodiment, each lipid nanoparticle vehicle comprises a differentcomposition of lipid components. In one embodiment, each lipidnanoparticle vehicle comprises3-Dimethylamino-2-(Cholest-5-en-3β-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), distearoylphosphatidylcholine (DSPC), Cholesterol,and1-[8′-(1,2-Dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethyleneglycol) (PEG-DMG). In one embodiment, each lipid nanoparticle vehiclefurther comprises Linoleyl alcohol. In another embodiment, the CLinDMA,DSPC, Cholesterol, PEG-DMG, and Linoleyl alcohol have a molar ratio ofabout 43/36/10/4/7. In another embodiment, the lipid nanoparticle hassize between 50 and 500 nm, or between 100 and 200 nm.

In one embodiment, the invention features a composition comprising alipid nanoparticle (LNP) vehicle having size between about 10 nm andabout 1000 nm, wherein: the vehicle further comprises one or morebiologically active molecules; the vehicle further comprises one or morecarrier molecules; and the lipid nanoparticle vehicle comprises acationic lipid, a neutral lipid, and a PEG-lipid. In one embodiment, thelipid nanoparticle vehicle comprises3-Dimethylamino-2-(Cholest-5-en-3β-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA), distearoylphosphatidylcholine (DSPC), Cholesterol, and1-[8′-(1,2-Dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethyleneglycol) (PEG-DMG). In one embodiment, the lipid nanoparticle vehiclefurther comprises Linoleyl alcohol. In another embodiment, the CLinDMA,DSPC, Cholesterol, PEG-DMG, and Linoleyl alcohol have a molar ratio ofabout 43/36/10/4/7. In another embodiment, the lipid nanoparticle hassize between 50 and 500 nm, or between 100 and 200 nm. In oneembodiment, the composition comprises at least two (e.g., 2, 3, 4, 5, 6,7, 8, 9, 10 or more) different biologically active molecules.

In one embodiment, the invention features a composition comprising alipid nanoparticle (LNP) vehicle having size between about 10 nm andabout 1000 nm, wherein: the vehicle further comprises one or morebiologically active molecules; the composition further comprises one ormore carrier molecules; and the lipid nanoparticle vehicle comprises acationic lipid, a neutral lipid, and a PEG-lipid. In one embodiment, thelipid nanoparticle vehicle comprises3-Dimethylamino-2-(Cholest-5-en-3-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA), distearoylphosphatidylcholine (DSPC), Cholesterol, and1-[8′-(1,2-Dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethyleneglycol) (PEG-DMG). In one embodiment, the lipid nanoparticle vehiclefurther comprises Linoleyl alcohol.

In another embodiment, the CLinDMA, DSPC, Cholesterol, PEG-DMG, andLinoleyl alcohol have a molar ratio of about 43/36/10/4/7. In anotherembodiment, the lipid nanoparticle has size between 50 and 500 nm, orbetween 100 and 200 nm. In one embodiment, the composition comprises atleast two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) differentbiologically active molecules.

In one embodiment, the invention features a composition comprising afirst lipid nanoparticle (LNP) vehicle and a second lipid nanoparticle(LNP) vehicle each having size between about 10 nm and 1000 nm, wherein:the first vehicle further comprises one or more short interferingnucleic acid (siNA) molecules comprising a sense strand and acomplementary antisense strand, each strand having between 15 and 30nucleotides in length, wherein the antisense strand comprises between 15and 30 nucleotides that are complementary to a mammalian RNA sequenceand the sense strand comprises between 15 and 30 nucleotides of saidmammalian RNA sequence; the second vehicle further comprises one or morecarrier molecules comprising nucleic acid sequence of at least 15nucleotides that is not complementary to said mammalian RNA sequence;and each vehicle comprises a cationic lipid, a neutral lipid, and aPEG-lipid. In one embodiment, each lipid nanoparticle vehicle comprisesthe same composition of lipid components. In another embodiment, eachlipid nanoparticle vehicle comprises a different composition of lipidcomponents. In one embodiment, each lipid nanoparticle vehicle comprises3-Dimethylamino-2-(Cholest-5-en-3β-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), distearoylphosphatidylcholine (DSPC), Cholesterol,and1-[8′-(1,2-Dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethyleneglycol) (PEG-DMG). In one embodiment, each lipid nanoparticle vehiclefurther comprises Linoleyl alcohol. In another embodiment, the CLinDMA,DSPC, Cholesterol, PEG-DMG, and Linoleyl alcohol have a molar ratio ofabout 43/36/10/4/7. In another embodiment, the lipid nanoparticle hassize between 50 and 500 nm, or between 100 and 200 nm. In oneembodiment, the composition comprises at least two (e.g., 2, 3, 4, 5, 6,7, 8, 9, 10 or more) different siNA molecules, for example as acocktail.

In one embodiment, the invention features a composition comprising alipid nanoparticle (LNP) vehicle having size between about 10 nm andabout 1000 nm, wherein: the vehicle further comprises one or more shortinterfering nucleic acid (siNA) molecules comprising a sense strand anda complementary antisense strand, each strand having between 15 and 30nucleotides in length, wherein the antisense strand comprises between 15and 30 nucleotides that are complementary to a mammalian RNA sequence,and the sense strand comprises between 15 and 30 nucleotides of saidmammalian RNA sequence; the vehicle further comprises one or morecarrier molecules comprising nucleic acid sequence of at least 15nucleotides that is not complementary to said mammalian RNA sequence;and the lipid nanoparticle vehicle comprises a cationic lipid, a neutrallipid, and a PEG-lipid. In one embodiment, the lipid nanoparticlevehicle comprises3-Dimethylamino-2-(Cholest-5-en-3β-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), distearoylphosphatidylcholine (DSPC), Cholesterol,and1-[8′-(1,2-Dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethyleneglycol) (PEG-DMG). In one embodiment, the lipid nanoparticle vehiclefurther comprises Linoleyl alcohol. In another embodiment, the CLinDMA,DSPC, Cholesterol, PEG-DMG, and Linoleyl alcohol have a molar ratio ofabout 43/36/10/4/7. In another embodiment, the lipid nanoparticle hassize between 50 and 500 nm, or between 100 and 200 nm. In oneembodiment, the composition comprises at least two (e.g., 2, 3, 4, 5, 6,7, 8, 9, 10 or more) different siNA molecules, for example as acocktail.

In one embodiment, the invention features a composition comprising alipid nanoparticle (LNP) vehicle having size between about 10 nm andabout 1000 nm, wherein: the vehicle further comprises one or more shortinterfering nucleic acid (siNA) molecules comprising a sense strand anda complementary antisense strand, each strand having between 15 and 30nucleotides in length, wherein the antisense strand comprises between 15and 30 nucleotides that are complementary to a mammalian RNA sequence,and the sense strand comprises between 15 and 30 nucleotides of saidmammalian RNA sequence; the composition further comprises one or morecarrier molecules comprising nucleic acid sequence of at least 15nucleotides that is not complementary to said mammalian RNA sequence;and the lipid nanoparticle vehicle comprises a cationic lipid, a neutrallipid, and a PEG-lipid. In one embodiment, the lipid nanoparticlevehicle comprises3-Dimethylamino-2-(Cholest-5-en-3β-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), distearoylphosphatidylcholine (DSPC), Cholesterol,and1-[8′-(1,2-Dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethyleneglycol) (PEG-DMG). In one embodiment, the lipid nanoparticle vehiclefurther comprises Linoleyl alcohol. In another embodiment, the CLinDMA,DSPC, Cholesterol, PEG-DMG, and Linoleyl alcohol have a molar ratio ofabout 43/36/10/4/7. In another embodiment, the lipid nanoparticle hassize between 50 and 500 nm, or between 100 and 200 nm. In oneembodiment, the composition comprises at least two (e.g., 2, 3, 4, 5, 6,7, 8, 9, 10 or more) different siNA molecules, for example as acocktail.

In one embodiment, the invention features a compound having Formula CLI:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or unsaturatedhydrocarbon, L is a linker, and R4 is cholesterol, a cholesterolderivative, a steroid hormone, or a bile acid. In one embodiment, R1 andR2 each independently is methyl, ethyl, propyl, isopropyl, or butyl. Inone embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, R4 is cholesterol. In one embodiment, L is a C1 to C10alkyl, alkyl ether, polyether, or polyethylene glycol linker. In anotherembodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (for example, monoester, diester), or succinyl linker.In one embodiment, R1 and R2 are methyl, R3 is linoyl, L is butyl, andR4 is cholesterol, which compound is generally referred to herein asCLinDMA or3-Dimethylamino-2-(Cholest-5-en-3β-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane.

In one embodiment, the invention features a compound having FormulaCLII:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or unsaturatedhydrocarbon, L is a linker, and R4 is cholesterol, a cholesterolderivative, a steroid hormone, or a bile acid. In one embodiment, R1 andR2 each independently is methyl, ethyl, propyl, isopropyl, or butyl. Inone embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, R4 is cholesterol. In one embodiment, L is a C1 to C10alkyl, alkyl ether, polyether, or polyethylene glycol linker. In anotherembodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (i.e., monoester, diester) or succinyl linker. In oneembodiment, R1 and R2 are methyl, R3 is linoyl, L is butyl, and R4 ischolesterol.

In one embodiment, the invention features a compound having FormulaCLIII:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or unsaturatedhydrocarbon, L is a linker, and R4 is cholesterol, a cholesterolderivative, a steroid hormone, or a bile acid. In one embodiment, R1 andR2 each independently is methyl, ethyl, propyl, isopropyl, or butyl. Inone embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, R4 is cholesterol. In one embodiment, L is a C1 to C10alkyl, alkyl ether, polyether, or polyethylene glycol linker. In oneembodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (i.e., monoester, diester), or succinyl linker. In oneembodiment, each R1 and R2 are methyl, R3 is linoyl, L is butyl, and R4is cholesterol.

In one embodiment, the invention features a compound having FormulaCLIV:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or unsaturatedhydrocarbon, L is a linker, and R4 is cholesterol, a cholesterolderivative, a steroid hormone, or a bile acid. In one embodiment, R1 andR2 each independently is methyl, ethyl, propyl, isopropyl, or butyl. Inone embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, R4 is cholesterol. In one embodiment, L is a C1 to C10alkyl, alkyl ether, polyether, or polyethylene glycol linker. In anotherembodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (i.e., monoester, diester), or succinyl linker. In oneembodiment, each R1 and R2 are methyl, R3 is linoyl, L is butyl, and R4is cholesterol.

In one embodiment, the invention features a compound having Formula CLV:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; and each R3 and R4 is independently a C12-C24aliphatic hydrocarbon, which can be the same or different. In oneembodiment, R1 and R2 each independently is methyl, ethyl, propyl,isopropyl, or butyl. In one embodiment, R3 and R4 each independently islinoyl, isostearyl, oleyl, elaidyl, petroselinyl, linolenyl,elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. In oneembodiment, R1 and R2 are methyl, and R3 and R4 are oleyl, this compoundis generally referred to herein as DMOBA orN,N-Dimethyl-3,4-dioleyloxybenzylamine.

In one embodiment, the invention features a compound having FormulaCLVI:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or unsaturatedhydrocarbon, L is a linker, and R4 is cholesterol, a cholesterolderivative, a steroid hormone, or a bile acid. In one embodiment, R1 andR2 each independently is methyl, ethyl, propyl, isopropyl, or butyl. Inone embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, R4 is cholesterol. In one embodiment, L is a C1 to C10alkyl, alkyl ether, polyether, or polyethylene glycol linker. In anotherembodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (i.e., monoester, diester), or succinyl linker. In oneembodiment, R1 and R2 are methyl, R3 is linoyl, L is butyl, and R4 ischolesterol.

In one embodiment, the invention features a compound having FormulaCLVII:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or unsaturatedhydrocarbon, L is a linker, and R4 is cholesterol, a cholesterolderivative, a steroid hormone, or a bile acid. In one embodiment, R1 andR2 each independently is methyl, ethyl, propyl, isopropyl, or butyl. Inone embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, R4 is cholesterol. In one embodiment, L is a C1 to C10alkyl, alkyl ether, polyether, or polyethylene glycol linker. In anotherembodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (i.e., monoester, diester), or succinyl linker. In oneembodiment, R1 and R2 are methyl, R3 is linoyl, L is butyl, and R4 ischolesterol.

In one embodiment, the invention features a compound having FormulaCLVIII:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; and each R3 and R4 is independently a C12-C24aliphatic hydrocarbon which can be the same or different. In oneembodiment, R1 and R2 each independently is methyl, ethyl, propyl,isopropyl, or butyl. In one embodiment, R3 and R4 each independently islinoyl, isostearyl, oleyl, elaidyl, petroselinyl, linolenyl,elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. In oneembodiment, each R1 and R2 are methyl, and R3 and R4 are linoyl.

In one embodiment, the invention features a compound having FormulaCLIX:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or unsaturatedhydrocarbon, L is a linker, and R4 is cholesterol, a cholesterolderivative, a steroid hormone, or a bile acid. In one embodiment, R1 andR2 each independently is methyl, ethyl, propyl, isopropyl, or butyl. Inone embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, R4 is cholesterol. In one embodiment, L is a C1 to C10alkyl, alkyl ether, polyether, or polyethylene glycol linker. In anotherembodiment, L is an acetal, amide, carbonyl, carbamate carbamide,carbamate, carbonate, ester (i.e., monoester, diester), or succinyllinker. In one embodiment, each R1 and R2 are methyl, R3 is linoyl, L isbutyl, and R4 is cholesterol.

In one embodiment, the invention features a compound having Formula CLX:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or unsaturatedhydrocarbon, L is a linker, and R4 is cholesterol, a cholesterolderivative, a steroid hormone, or a bile acid. In one embodiment, R1 andR2 each independently is methyl, ethyl, propyl, isopropyl, or butyl. Inone embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, R4 is cholesterol. In one embodiment, L is a C1 to C10alkyl, alkyl ether, polyether, or polyethylene glycol linker. In anotherembodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (i.e., monoester, diester), or succinyl linker. In oneembodiment, each R1 and R2 are methyl, R3 is linoyl, L is butyl, and R4is cholesterol.

In one embodiment, the invention features a compound having FormulaCLXI:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or unsaturatedhydrocarbon, L is a linker, and R4 is cholesterol, a cholesterolderivative, a steroid hormone, or a bile acid. In one embodiment, R1 andR2 each independently is methyl, ethyl, propyl, isopropyl, or butyl. Inone embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, R4 is cholesterol. In one embodiment, L is a C1 to C10alkyl, alkyl ether, polyether, or polyethylene glycol linker. In anotherembodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (i.e., monoester, diester), or succinyl linker. In oneembodiment, each R1 and R2 are methyl, R3 is linoyl, L is butyl, and R4is cholesterol.

In one embodiment, the invention features a compound having FormulaCLXIIa or CLXIIb:

wherein R0 and each R1 and R2 is independently a C1 to C10 alkyl,alkynyl, or aryl hydrocarbon; R3 is a C9-C24 aliphatic saturated orunsaturated hydrocarbon, L is a linker, and R4 is cholesterol, acholesterol derivative, a steroid hormone, or a bile acid. In oneembodiment, R1 and R2 each independently is methyl, ethyl, propyl,isopropyl, or butyl. In one embodiment, R3 is linoyl, isostearyl, oleyl,elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,palmitoyl, or lauroyl. In one embodiment, R4 is cholesterol. In oneembodiment, L is a C1 to C10 alkyl, alkyl ether, polyether, orpolyethylene glycol linker. In another embodiment, L is an acetal,amide, carbonyl, carbamide, carbamate, carbonate, ester (i.e.,monoester, diester), or succinyl linker. In one embodiment, each R1 andR2 are methyl, R3 is linoyl, L is butyl, and R4 is cholesterol.

In one embodiment, the invention features a compound having FormulaCLXIII:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or unsaturatedhydrocarbon, L is a linker, and R4 is cholesterol, a cholesterolderivative, a steroid hormone, or a bile acid. In one embodiment, R1 andR2 each independently is methyl, ethyl, propyl, isopropyl, or butyl. Inone embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, R4 is cholesterol. In one embodiment, L is a C1 to C10alkyl, alkyl ether, polyether, or polyethylene glycol linker. In anotherembodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (i.e., monoester, diester), or succinyl linker. In oneembodiment, each R1 and R2 are methyl, R3 is linoyl, L is butyl, and R4is cholesterol.

In one embodiment, the invention features a compound having FormulaCLXIVa and CLXIVb:

wherein R0 and each R1 and R2 is independently a C1 to C10 alkyl,alkynyl, or aryl hydrocarbon; R3 is a C9-C24 aliphatic saturated orunsaturated hydrocarbon, L is a linker, and R4 is cholesterol, acholesterol derivative, a steroid hormone, or a bile acid. In oneembodiment, R1 and R2 each independently is methyl, ethyl, propyl,isopropyl, or butyl. In one embodiment, R3 is linoyl, isostearyl, oleyl,elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,palmitoyl, or lauroyl. In one embodiment, R4 is cholesterol. In oneembodiment, L is a C1 to C10 alkyl, alkyl ether, polyether, orpolyethylene glycol linker. In another embodiment, L is an acetal,amide, carbonyl, carbamide, carbamate, carbonate, ester (i.e.,monoester, diester), or succinyl linker. In one embodiment, each R1 andR2 are methyl, R3 is linoyl, L is butyl, and R4 is cholesterol.

In one embodiment, the invention features a compound having FormulaCLXV:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; L is a linker, and each R3 is independentlycholesterol, a cholesterol derivative, a steroid hormone, or a bileacid. In one embodiment, R1 and R2 each independently is methyl, ethyl,propyl, isopropyl, or butyl. In one embodiment, R3 is cholesterol. Inone embodiment, L is a C1 to C10 alkyl, alkyl ether, polyether, orpolyethylene glycol linker. In another embodiment, L is an acetal,amide, carbonyl, carbamide, carbamate, carbonate, ester (i.e.,monoester, diester), or succinyl linker. In one embodiment, each R1 andR2 are methyl, R3 is cholesterol, and L is butyl.

In one embodiment, the invention features a compound having FormulaCLXVI:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; each L is a linker whose structure is independent ofthe other L, and each R3 is independently cholesterol, a cholesterolderivative, a steroid hormone, or a bile acid. In one embodiment, R1 andR2 each independently is methyl, ethyl, propyl, isopropyl, or butyl. Inone embodiment, R3 is cholesterol. In one embodiment, L is a C1 to C10alkyl, alkyl ether, polyether, or polyethylene glycol linker. In anotherembodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (i.e., monoester, diester), or succinyl linker. In oneembodiment, each R1 and R2 are methyl, R3 is cholesterol, and L isbutyl.

In one embodiment, the invention features a compound having FormulaCLXVII:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon and R3 is a C9-C24 aliphatic saturated or unsaturatedhydrocarbon. In one embodiment, R1 and R2 each independently is methyl,ethyl, propyl, isopropyl, or butyl. In one embodiment, R3 is linoyl,isostearyl, oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl,arachidyl, myristoyl, palmitoyl, or lauroyl. In one embodiment, each R1and R2 are methyl and R3 is linoyl.

In one embodiment, the invention features a compound having FormulaCLXVIII:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or unsaturatedhydrocarbon. In one embodiment, R1 and R2 each independently is methyl,ethyl, propyl, isopropyl, or butyl. In one embodiment, R3 is linoyl,isostearyl, oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl,arachidyl, myristoyl, palmitoyl, or lauroyl. In one embodiment, each R1and R2 are methyl and R3 is linoyl.

In one embodiment, the invention features a compound having FormulaCLXIX:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 and R4 are each individually a C9-C24 aliphaticsaturated or unsaturated hydrocarbon, which can be the same ordifferent. In one embodiment, R1 and R2 each independently is methyl,ethyl, propyl, isopropyl, or butyl. In one embodiment, R3 and R4 eachindividually is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, R3 or R4 is cholesterol, a cholesterol derivative, asteroid hormone, or a bile acid.

In one embodiment, the invention features a compound having FormulaCLXX:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 and R4 are each individually a C9-C24 aliphaticsaturated or unsaturated hydrocarbon, which can be the same ordifferent. In one embodiment, R1 and R2 each independently is methyl,ethyl, propyl, isopropyl, or butyl. In one embodiment, R3 and R4 eachindividually is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, R3 or R4 is cholesterol, a cholesterol derivative, asteroid hormone, or a bile acid.

In one embodiment, the invention features a compound having FormulaCLXXI:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 and R4 are each individually a C9-C24 aliphaticsaturated or unsaturated hydrocarbon, which can be the same ordifferent. In one embodiment, R1 and R2 each independently is methyl,ethyl, propyl, isopropyl, or butyl. In one embodiment, R3 and R4 eachindividually is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, R3 or R4 is cholesterol, a cholesterol derivative, asteroid hormone, or a bile acid.

In one embodiment, the invention features a compound having FormulaCLXXII:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 and R4 are each individually a C9-C24 aliphaticsaturated or unsaturated hydrocarbon, which can be the same ordifferent. In one embodiment, R1 and R2 each independently is methyl,ethyl, propyl, isopropyl, or butyl. In one embodiment, R3 and R4 eachindividually is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, R3 or R4 is cholesterol, a cholesterol derivative, asteroid hormone, or a bile acid.

In one embodiment, the invention features a compound having FormulaCLXXIII:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or unsaturatedhydrocarbon, and L is a linker. In one embodiment, R1 and R2 eachindependently is methyl, ethyl, propyl, isopropyl, or butyl. In oneembodiment, R3 and R4 each individually is linoyl, isostearyl, oleyl,elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,palmitoyl, or lauroyl. In one embodiment, R3 or R4 is cholesterol, acholesterol derivative, a steroid hormone, or a bile acid. In oneembodiment, L is a C1 to C10 alkyl, alkyl ether, polyether, orpolyethylene glycol linker. In another embodiment, L is an acetal,amide, carbonyl, carbamide, carbamate, carbonate, ester (i.e.,monoester, diester), or succinyl linker.

In one embodiment, the invention features a compound having FormulaCLXXIV:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or unsaturatedhydrocarbon, and L is a linker. In one embodiment, R1 and R2 eachindependently is methyl, ethyl, propyl, isopropyl, or butyl. In oneembodiment, R3 and R4 each individually is linoyl, isostearyl, oleyl,elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,palmitoyl, or lauroyl. In one embodiment, R3 or R4 is cholesterol, acholesterol derivative, a steroid hormone, or a bile acid. In oneembodiment, L is a C1 to C10 alkyl, alkyl ether, polyether, orpolyethylene glycol linker. In another embodiment, L is an acetal,amide, carbonyl, carbamide, carbamate, carbonate, ester (i.e.,monoester, diester), or succinyl linker.

In one embodiment, the invention features a compound having FormulaCLXXV:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or unsaturatedhydrocarbon, and L is a linker. In one embodiment, R1 and R2 eachindependently is methyl, ethyl, propyl, isopropyl, or butyl. In oneembodiment, R3 and R4 each individually is linoyl, isostearyl, oleyl,elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,palmitoyl, or lauroyl. In one embodiment, R3 or R4 is cholesterol, acholesterol derivative, a steroid hormone, or a bile acid. In oneembodiment, L is a C1 to C10 alkyl, alkyl ether, polyether, orpolyethylene glycol linker. In another embodiment, L is an acetal,amide, carbonyl, carbamide, carbamate, carbonate, ester (i.e.,monoester, diester), or succinyl linker.

In one embodiment, the invention features a compound having FormulaCLXXVI:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 and R4 are each individually a C9-C24 aliphaticsaturated or unsaturated hydrocarbon, which can be the same ordifferent. In one embodiment, R1 and R2 each independently is methyl,ethyl, propyl, isopropyl, or butyl. In one embodiment, R3 and R4 eachindividually is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, R3 or R4 is cholesterol, a cholesterol derivative, asteroid hormone, or a bile acid.

In one embodiment, the invention features a compound having FormulaCLXXVII:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 and R4 are each individually a C9-C24 aliphaticsaturated or unsaturated hydrocarbon, and L is a linker. In oneembodiment, R1 and R2 each independently is methyl, ethyl, propyl,isopropyl, or butyl. In one embodiment, R3 and R4 each individually islinoyl, isostearyl, oleyl, elaidyl, petroselinyl, linolenyl,elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. In oneembodiment, R3 or R4 is cholesterol, a cholesterol derivative, a steroidhormone, or a bile acid. In one embodiment, L is a C1 to C10 alkyl,alkyl ether, polyether, or polyethylene glycol linker. In anotherembodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (i.e., monoester, diester), or succinyl linker.

In one embodiment, the invention features a compound having FormulaCLXXVIII:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 and R4 are each individually a C9-C24 aliphaticsaturated or unsaturated hydrocarbon, which can be the same ordifferent. In one embodiment, R1 and R2 each independently is methyl,ethyl, propyl, isopropyl, or butyl. In one embodiment, R3 and R4 eachindividually is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, R3 or R4 is cholesterol, a cholesterol derivative, asteroid hormone, or a bile acid.

In one embodiment, the invention features a compound having FormulaCLXXIX:

wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl, oraryl hydrocarbon; R3 and R4 are each individually a C9-C24 aliphaticsaturated or unsaturated hydrocarbon, which can be the same ordifferent. In one embodiment, R1 and R2 each independently is methyl,ethyl, propyl, isopropyl, or butyl. In one embodiment, R3 and R4 eachindividually is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, R3 or R4 is cholesterol, a cholesterol derivative, asteroid hormone, or a bile acid.

In one embodiment, the invention features a compound having FormulaCLXXX:

wherein R3 is a C9-C24 aliphatic saturated or unsaturated hydrocarbon,each L is independently a linker, and R4 is cholesterol, a cholesterolderivative, a steroid hormone, or a bile acid. In one embodiment, R3 islinoyl, isostearyl, oleyl, elaidyl, petroselinyl, linolenyl,elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. In oneembodiment, R4 is cholesterol. In one embodiment, each L isindependently a C1 to C10 alkyl, alkyl ether, polyether, or polyethyleneglycol linker with or without a disulphide linkage. In anotherembodiment, each L is independently an acetal, amide, carbonyl,carbamide, carbamate, carbonate, ester (for example, monoester,diester), or succinyl linker. In one embodiment, R3 is linoyl and R4 ischolesterol.

In one embodiment, the invention features a compound having FormulaCLXXXI:

wherein R3 is a C9-C24 aliphatic saturated or unsaturated hydrocarbon,each L is independently a linker, and R4 is cholesterol, a cholesterolderivative, a steroid hormone, or a bile acid. In one embodiment, R3 islinoyl, isostearyl, oleyl, elaidyl, petroselinyl, linolenyl,elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. In oneembodiment, R4 is cholesterol. In one embodiment, each L isindependently a C1 to C10 alkyl, alkyl ether, polyether, or polyethyleneglycol linker with or without a disulphide linkage. In anotherembodiment, each L is independently an acetal, amide, carbonyl,carbamide, carbamate, carbonate, ester (i.e., monoester, diester) orsuccinyl linker. In one embodiment, R3 is linoyl, L is butyl, and R4 ischolesterol.

In one embodiment, the invention features a compound having FormulaCLXXXII:

wherein each R1, R2 and R5 is independently hydrogen, methyl, ethyl,propyl, isopropyl, or butyl, R3 is a C9-C24 aliphatic saturated orunsaturated hydrocarbon, each L is independently a linker, and R4 ischolesterol, a cholesterol derivative, a steroid hormone, or a bileacid. In one embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl,petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl,or lauroyl. In one embodiment, R4 is cholesterol. In one embodiment,each L is independently a C1 to C10 alkyl, alkyl ether, polyether, orpolyethylene glycol linker with or without a disulphide linkage. In oneembodiment, each L is independently an acetal, amide, carbonyl,carbamide, carbamate, carbonate, ester (i.e., monoester, diester), orsuccinyl linker. In one embodiment, R3 is linoyl and R4 is cholesterol.

In one embodiment, the invention features a compound having FormulaCLXXXIII:

wherein each R1, R2 and R5 is independently hydrogen, methyl, ethyl,propyl, isopropyl, or butyl, R3 is a C9-C24 aliphatic saturated orunsaturated hydrocarbon, each L is independently a linker, and R4 ischolesterol, a cholesterol derivative, a steroid hormone, or a bileacid. In one embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl,petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl,or lauroyl. In one embodiment, R4 is cholesterol. In one embodiment,each L is independently a C1 to C10 alkyl, alkyl ether, polyether, orpolyethylene glycol linker with or without a disulphide linkage. Inanother embodiment, each L is independently an acetal, amide, carbonyl,carbamide, carbamate, carbonate, ester (i.e., monoester, diester), orsuccinyl linker. In one embodiment, R3 is linoyl and R4 is cholesterol.

In one embodiment, the invention features a compound having FormulaCLXXXIV:

wherein each R1 and R2 is independently hydrogen, methyl, ethyl, propyl,isopropyl, or butyl, each R3 and R4 is independently a C12-C24 aliphatichydrocarbon, which can be the same or different, and each L isindependently a linker, which can be present or absent. In oneembodiment, R3 and R4 each independently is linoyl, isostearyl, oleyl,elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,palmitoyl, or lauroyl. In one embodiment, each L is independently a C1to C10 alkyl, alkyl ether, polyether, or polyethylene glycol linker withor without a disulphide linkage. In another embodiment, each L isindependently an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (i.e., monoester, diester), or succinyl linker. In oneembodiment, R3 and R4 are oleyl.

In one embodiment, each R1 and R2 is independently hydrogen, methyl,ethyl, propyl, isopropyl, or butyl, R3 is a C9-C24 aliphatic saturatedor unsaturated hydrocarbon, each L is independently a linker, and R4 ischolesterol, a cholesterol derivative, a steroid hormone, or a bileacid. In one embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl,petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl,or lauroyl. In one embodiment, R4 is cholesterol. In one embodiment,each L is independently a C1 to C10 alkyl, alkyl ether, polyether, orpolyethylene glycol linker with or without a disulphide linkage. Inanother embodiment, each L is independently an acetal, amide, carbonyl,carbamide, carbamate, carbonate, ester (i.e., monoester, diester), orsuccinyl linker. In one embodiment, R3 is linoyl, L is butyl, and R4 ischolesterol.

In one embodiment, the invention features a compound having FormulaCLXXXV:

wherein each R1 and R2 is independently hydrogen, methyl, ethyl, propyl,isopropyl, or butyl, each R3 and R4 is independently a C12-C24 aliphatichydrocarbon, which can be the same or different, and each L isindependently a linker, which can be present or absent. In oneembodiment, R3 and R4 each independently is linoyl, isostearyl, oleyl,elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,palmitoyl, or lauroyl. In one embodiment, each L is independently a C1to C10 alkyl, alkyl ether, polyether, or polyethylene glycol linker withor without a disulphide linkage. In another embodiment, each L isindependently an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (i.e., monoester, diester), or succinyl linker. In oneembodiment, R3 and R4 are oleyl.

In one embodiment, each R1 and R2 is independently hydrogen, methyl,ethyl, propyl, isopropyl, or butyl, R3 is a C9-C24 aliphatic saturatedor unsaturated hydrocarbon, each L is independently a linker, and R4 ischolesterol, a cholesterol derivative, a steroid hormone, or a bileacid. In one embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl,petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl,or lauroyl. In one embodiment, R4 is cholesterol. In one embodiment,each L is independently a C1 to C10 alkyl, alkyl ether, polyether, orpolyethylene glycol linker with or without a disulphide linkage. Inanother embodiment, each L is independently an acetal, amide, carbonyl,carbamide, carbamate, carbonate, ester (i.e., monoester, diester), orsuccinyl linker. In one embodiment, R3 is linoyl, L is butyl, and R4 ischolesterol.

In one embodiment, the invention features a compound having FormulaCLXXXVI:

wherein each R1 and R2 is independently hydrogen, methyl, ethyl, propyl,isopropyl, or butyl, each R3 and R4 is independently a C12-C24 aliphatichydrocarbon, which can be the same or different, and each L isindependently a linker, which can be present or absent. In oneembodiment, R3 and R4 each independently is linoyl, isostearyl, oleyl,elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,palmitoyl, or lauroyl. In one embodiment, each L is independently a C1to C10 alkyl, alkyl ether, polyether, or polyethylene glycol linker withor without a disulphide linkage. In another embodiment, each L isindependently an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (i.e., monoester, diester), or succinyl linker. In oneembodiment, R3 and R4 are oleyl.

In one embodiment, each R1 and R2 is independently hydrogen, methyl,ethyl, propyl, isopropyl, or butyl, R3 is a C9-C24 aliphatic saturatedor unsaturated hydrocarbon, each L is independently a linker, and R4 ischolesterol, a cholesterol derivative, a steroid hormone, or a bileacid. In one embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl,petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl,or lauroyl. In one embodiment, R4 is cholesterol. In one embodiment,each L is independently a C1 to C10 alkyl, alkyl ether, polyether, orpolyethylene glycol linker with or without a disulphide linkage. Inanother embodiment, each L is independently an acetal, amide, carbonyl,carbamide, carbamate, carbonate, ester (i.e., monoester, diester), orsuccinyl linker. In one embodiment, R3 is linoyl, L is butyl, and R4 ischolesterol.

In one embodiment, the invention features a compound having FormulaCLXXXVII:

wherein each R1 and R2 is independently hydrogen, methyl, ethyl, propyl,isopropyl, or butyl, each R3 and R4 is independently a C12-C24 aliphatichydrocarbon, which can be the same or different, and each L isindependently a linker, which can be present or absent. In oneembodiment, R3 and R4 each independently is linoyl, isostearyl, oleyl,elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,palmitoyl, or lauroyl. In one embodiment, each L is independently a C1to C10 alkyl, alkyl ether, polyether, or polyethylene glycol linker withor without a disulphide linkage. In another embodiment, each L isindependently an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (i.e., monoester, diester), or succinyl linker. In oneembodiment, R3 and R4 are oleyl.

In one embodiment, each R1 and R2 is independently hydrogen, methyl,ethyl, propyl, isopropyl, or butyl, R3 is a C9-C24 aliphatic saturatedor unsaturated hydrocarbon, each L is independently a linker, and R4 ischolesterol, a cholesterol derivative, a steroid hormone, or a bileacid. In one embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl,petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl,or lauroyl. In one embodiment, R4 is cholesterol. In one embodiment,each L is independently a C1 to C10 alkyl, alkyl ether, polyether, orpolyethylene glycol linker with or without a disulphide linkage. Inanother embodiment, each L is independently an acetal, amide, carbonyl,carbamide, carbamate, carbonate, ester (i.e., monoester, diester), orsuccinyl linker. In one embodiment, R3 is linoyl, L is butyl, and R4 ischolesterol.

In one embodiment, the invention features a compound having FormulaCLXXXVIII:

wherein each R1 and R2 is independently hydrogen, methyl, ethyl, propyl,isopropyl, or butyl, each R3 and R4 is independently a C12-C24 aliphatichydrocarbon, which can be the same or different, and each L isindependently a linker, which can be present or absent. In oneembodiment, R3 and R4 each independently is linoyl, isostearyl, oleyl,elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,palmitoyl, or lauroyl. In one embodiment, each L is independently a C1to C10 alkyl, alkyl ether, polyether, or polyethylene glycol linker withor without a disulphide linkage. In another embodiment, each L isindependently an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (i.e., monoester, diester), or succinyl linker. In oneembodiment, R3 and R4 are oleyl.

In one embodiment, each R1 and R2 is independently hydrogen, methyl,ethyl, propyl, isopropyl, or butyl, R3 is a C9-C24 aliphatic saturatedor unsaturated hydrocarbon, each L is independently a linker, and R4 ischolesterol, a cholesterol derivative, a steroid hormone, or a bileacid. In one embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl,petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl,or lauroyl. In one embodiment, R4 is cholesterol. In one embodiment,each L is independently a C1 to C10 alkyl, alkyl ether, polyether, orpolyethylene glycol linker with or without a disulphide linkage. Inanother embodiment, each L is independently an acetal, amide, carbonyl,carbamide, carbamate, carbonate, ester (i.e., monoester, diester), orsuccinyl linker. In one embodiment, R3 is linoyl, L is butyl, and R4 ischolesterol.

In one embodiment, the invention features a compound having FormulaCLXXXIX:

wherein each R1 and R2 is independently hydrogen, methyl, ethyl, propyl,isopropyl, or butyl, each R3 and R4 is independently a C12-C24 aliphatichydrocarbon, which can be the same or different, and each L isindependently a linker, which can be present or absent. In oneembodiment, R3 and R4 each independently is linoyl, isostearyl, oleyl,elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,palmitoyl, or lauroyl. In one embodiment, each L is independently a C1to C10 alkyl, alkyl ether, polyether, or polyethylene glycol linker withor without a disulphide linkage. In another embodiment, each L isindependently an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (i.e., monoester, diester), or succinyl linker. In oneembodiment, R3 and R4 are oleyl.

In one embodiment, each R1 and R2 is independently hydrogen, methyl,ethyl, propyl, isopropyl, or butyl, R3 is a C9-C24 aliphatic saturatedor unsaturated hydrocarbon, each L is independently a linker, and R4 ischolesterol, a cholesterol derivative, a steroid hormone, or a bileacid. In one embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl,petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl,or lauroyl. In one embodiment, R4 is cholesterol. In one embodiment,each L is independently a C1 to C10 alkyl, alkyl ether, polyether, orpolyethylene glycol linker with or without a disulphide linkage. Inanother embodiment, each L is independently an acetal, amide, carbonyl,carbamide, carbamate, carbonate, ester (i.e., monoester, diester), orsuccinyl linker. In one embodiment, R3 is linoyl, L is butyl, and R4 ischolesterol.

In one embodiment, the invention features a compound having FormulaCLXXXX:

wherein each R1 and R2 is independently hydrogen, methyl, ethyl, propyl,isopropyl, or butyl, each R3 and R4 is independently a C12-C24 aliphatichydrocarbon, which can be the same or different, and each L isindependently a linker, which can be present or absent. In oneembodiment, R3 and R4 each independently is linoyl, isostearyl, oleyl,elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,palmitoyl, or lauroyl. In one embodiment, each L is independently a C1to C10 alkyl, alkyl ether, polyether, or polyethylene glycol linker withor without a disulphide linkage. In another embodiment, each L isindependently an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (i.e., monoester, diester), or succinyl linker. In oneembodiment, R3 and R4 are oleyl.

In one embodiment, each R1 and R2 is independently hydrogen, methyl,ethyl, propyl, isopropyl, or butyl, R3 is a C9-C24 aliphatic saturatedor unsaturated hydrocarbon, each L is independently a linker, and R4 ischolesterol, a cholesterol derivative, a steroid hormone, or a bileacid. In one embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl,petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl,or lauroyl. In one embodiment, R4 is cholesterol. In one embodiment,each L is independently a C1 to C10 alkyl, alkyl ether, polyether, orpolyethylene glycol linker with or without a disulphide linkage. Inanother embodiment, each L is independently an acetal, amide, carbonyl,carbamide, carbamate, carbonate, ester (i.e., monoester, diester), orsuccinyl linker. In one embodiment, R3 is linoyl, L is butyl, and R4 ischolesterol.

In one embodiment, the invention features a compound having FormulaCLXXXXI:

wherein each R1 and R2 is independently hydrogen, methyl, ethyl, propyl,isopropyl, or butyl, each R3 and R4 is independently a C12-C24 aliphatichydrocarbon, which can be the same or different, and each L isindependently a linker, which can be present or absent. In oneembodiment, R3 and R4 each independently is linoyl, isostearyl, oleyl,elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,palmitoyl, or lauroyl. In one embodiment, each L is independently a C1to C10 alkyl, alkyl ether, polyether, or polyethylene glycol linker withor without a disulphide linkage. In another embodiment, each L isindependently an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (i.e., monoester, diester), or succinyl linker. In oneembodiment, R3 and R4 are oleyl.

In one embodiment, each R1 and R2 is independently hydrogen, methyl,ethyl, propyl, isopropyl, or butyl, R3 is a C9-C24 aliphatic saturatedor unsaturated hydrocarbon, each L is independently a linker, and R4 ischolesterol, a cholesterol derivative, a steroid hormone, or a bileacid. In one embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl,petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl,or lauroyl. In one embodiment, R4 is cholesterol. In one embodiment,each L is independently a C1 to C10 alkyl, alkyl ether, polyether, orpolyethylene glycol linker with or without a disulphide linkage. Inanother embodiment, each L is independently an acetal, amide, carbonyl,carbamide, carbamate, carbonate, ester (i.e., monoester, diester), orsuccinyl linker. In one embodiment, R3 is linoyl, L is butyl, and R4 ischolesterol.

In one embodiment, the invention features a compound having FormulaCLXXXXII:

wherein R3 is a C9-C24 aliphatic saturated or unsaturated hydrocarbon, Lis a linker, and R4 is cholesterol, a cholesterol derivative, a steroidhormone, or a bile acid. In one embodiment, R3 is linoyl, isostearyl,oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl,myristoyl, palmitoyl, or lauroyl. In one embodiment, R4 is cholesterol.In one embodiment, L is a C1 to C10 alkyl, alkyl ether, polyether, orpolyethylene glycol linker. In another embodiment, L is an acetal,amide, carbonyl, carbamide, carbamate, carbonate, ester (i.e.,monoester, diester), or succinyl linker. In one embodiment, R3 islinoyl, L is butyl, and R4 is cholesterol.

In one embodiment, the invention features a compound having FormulaCLXXXXIII:

wherein each R3 and R4 is independently a C8-C24 aliphatic hydrocarbon,which can be the same or different, and each L is independently alinker, which can be present or absent. In one embodiment, R3 and R4each independently is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, each L is independently a C1 to C10 alkyl, alkyl ether,polyether, or polyethylene glycol linker with or without a disulphidelinkage. In another embodiment, each L is independently an acetal,amide, carbonyl, carbamide, carbamate, carbonate, ester (i.e.,monoester, diester), ether, or succinyl linker. In one embodiment, R3and R4 are dodecyl (C12). In one embodiment, R3 and R4 are oleyl.

In one embodiment, the invention features a compound having FormulaCLXXXXIV:

wherein each R3 and R4 is independently a C8-C24 aliphatic hydrocarbon,which can be the same or different, and each L is independently alinker, which can be present or absent. In one embodiment, R3 and R4each independently is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, each L is independently a C1 to C10 alkyl, alkyl ether,polyether, or polyethylene glycol linker with or without a disulphidelinkage. In another embodiment, each L is independently an acetal,amide, carbonyl, carbamide, carbamate, carbonate, ester (i.e.,monoester, diester), ether, or succinyl linker. In one embodiment, R3and R4 are dodecyl (C12). In one embodiment, R3 and R4 are oleyl.

In one embodiment, the invention features a compound having FormulaCLXXXXV:

wherein each R3 and R4 is independently a C8-C24 aliphatic hydrocarbon,which can be the same or different, and each L is independently alinker, which can be present or absent. In one embodiment, R3 and R4each independently is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, each L is independently a C1 to C10 alkyl, alkyl ether,polyether, or polyethylene glycol linker with or without a disulphidelinkage. In another embodiment, each L is independently an acetal,amide, carbonyl, carbamide, carbamate, carbonate, ester (i.e.,monoester, diester), ether, or succinyl linker. In one embodiment, R3and R4 are dodecyl (C12). In one embodiment, R3 and R4 are oleyl.

In one embodiment, the invention features a compound having FormulaCLXXXXVI:

wherein each R3 and R4 is independently a C8-C24 aliphatic hydrocarbon,which can be the same or different, and each L is independently alinker, which can be present or absent. In one embodiment, R3 and R4each independently is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, each L is independently a C1 to C10 alkyl, alkyl ether,polyether, or polyethylene glycol linker with or without a disulphidelinkage. In another embodiment, each L is independently an acetal,amide, carbonyl, carbamide, carbamate, carbonate, ester (i.e.,monoester, diester), ether, or succinyl linker. In one embodiment, R3and R4 are dodecyl (C12). In one embodiment, R3 and R4 are oleyl.

In one embodiment, any of compounds CLI-CLXXXXVI include a biodegradablelinkage as L, for example a disulphide linkage such as:

In one embodiment, the invention features a compound having Formula NLI:

wherein R1 is H, OH, or a C1 to C10 alkyl, alkynyl, or aryl hydrocarbonor alcohol; R3 is a C9-C24 aliphatic saturated or unsaturatedhydrocarbon, L is a linker, and R4 is cholesterol, a cholesterolderivative, a steroid hormone, or a bile acid. In one embodiment, R1 isOH, methyl, ethyl, propyl, isopropyl, or butyl or its correspondingalcohol. In one embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl,petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl,or lauroyl. In one embodiment, R4 is cholesterol. In one embodiment, Lis a C1 to C10 alkyl, alkyl ether, polyether, or polyethylene glycollinker. In another embodiment, L is an acetal, amide, carbonyl,carbamide, carbamate, carbonate, ester (for example, monoester,diester), or succinyl linker. In one embodiment, R1 is OH, R3 is linoyl,L is butyl, and R4 is cholesterol.

In one embodiment, the invention features a compound having FormulaNLII:

wherein R1 is H, OH, or a C1 to C10 alkyl, alkynyl, or aryl hydrocarbonor alcohol; R3 is a C9-C24 aliphatic saturated or unsaturatedhydrocarbon, L is a linker, and R4 is cholesterol, a cholesterolderivative, a steroid hormone, or a bile acid. In one embodiment, R1 ismethyl, ethyl, propyl, isopropyl, or butyl or its corresponding alcohol.In one embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl,petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl,or lauroyl. In one embodiment, R4 is cholesterol. In one embodiment, Lis a C1 to C10 alkyl, alkyl ether, polyether, or polyethylene glycollinker. In another embodiment, L is an acetal, amide, carbonyl,carbamide, carbamate, carbonate, ester (i.e., monoester, diester) orsuccinyl linker. In one embodiment, R1 is OH, R3 is linoyl, L is butyl,and R4 is cholesterol.

In one embodiment, the invention features a compound having FormulaNLIII:

wherein R1 is H, OH, a C1 to C10 alkyl, alkynyl, or aryl hydrocarbon oralcohol; and each R3 and R4 is independently a C12-C24 aliphatichydrocarbon, which can be the same or different. In one embodiment, R1is methyl, ethyl, propyl, isopropyl, or butyl or its correspondingalcohol. In one embodiment, R3 and R4 each independently is linoyl,isostearyl, oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl,arachidyl, myristoyl, palmitoyl, or lauroyl. In one embodiment, R1 isOH, and R3 and R4 are oleyl, this compound is generally referred toherein as DOBA or dioleyloxybenzyl alcohol.

In one embodiment, the invention features a compound having FormulaNLIV:

wherein R1 is H, OH a C1 to C10 alkyl, alkynyl, or aryl hydrocarbon oralcohol; R3 is a C9-C24 aliphatic saturated or unsaturated hydrocarbon,L is a linker, and R4 is cholesterol, a cholesterol derivative, asteroid hormone, or a bile acid. In one embodiment, R1 is methyl, ethyl,propyl, isopropyl, or butyl or its corresponding alcohol. In oneembodiment, R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, R4 is cholesterol. In one embodiment, L is a C1 to C10alkyl, alkyl ether, polyether, or polyethylene glycol linker. In anotherembodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (i.e., monoester, diester), or succinyl linker. In oneembodiment, R1 is OH, R3 is linoyl, L is butyl, and R4 is cholesterol.

In one embodiment, the invention features a compound having Formula NLV:

wherein R1 is H, OH a C1 to C10 alkyl, alkynyl, or aryl hydrocarbon oralcohol; R3 is a C9-C24 aliphatic saturated or unsaturated hydrocarbon,L is a linker, and R4 is cholesterol, a cholesterol derivative, asteroid hormone, or a bile acid. In one embodiment, R1 is methyl, ethyl,propyl, isopropyl, or butyl or its corresponding alcohol. In oneembodiment, R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, R4 is cholesterol. In one embodiment, L is a C1 to C10alkyl, alkyl ether, polyether, or polyethylene glycol linker. In anotherembodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,carbonate, ester (i.e., monoester, diester), or succinyl linker. In oneembodiment, R1 is OH, R3 is linoyl, L is butyl, and R4 is cholesterol.

In one embodiment, the invention features a compound having FormulaNLVI:

wherein R1 is H, OH, a C1 to C10 alkyl, alkynyl, or aryl hydrocarbon oralcohol; R3 is a C9-C24 aliphatic saturated or unsaturated hydrocarbon,and each L is a linker. In one embodiment, R1 is methyl, ethyl, propyl,isopropyl, or butyl or its corresponding alcohol. In one embodiment, R3and R4 each individually is linoyl, isostearyl, oleyl, elaidyl,petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl,or lauroyl. In one embodiment, R3 or R4 is cholesterol, a cholesterolderivative, a steroid hormone, or a bile acid. In one embodiment, each Lindependently is a C1 to C10 alkyl, alkyl ether, polyether, orpolyethylene glycol linker. In another embodiment, each L independentlyis an acetal, amide, carbonyl, carbamide, carbamate, carbonate, ester(i.e., monoester, diester), or succinyl linker.

In one embodiment, the invention features a compound having FormulaNLVII:

wherein R1 is independently H, OH, a C1 to C10 alkyl, alkynyl, or arylhydrocarbon or alcohol; R3 and R4 are each individually a C9-C24aliphatic saturated or unsaturated hydrocarbon, which can be the same ordifferent. In one embodiment, R1 is methyl, ethyl, propyl, isopropyl, orbutyl or its corresponding alcohol. In one embodiment, R3 and R4 eachindividually is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. Inone embodiment, R3 or R4 is cholesterol, a cholesterol derivative, asteroid hormone, or a bile acid.

In one embodiment, each O—R3 and/or O—R4 of any compound having FormulaeCLI-CLXIV, CLXVII-CLXXII, CLXXVI, and CLXXVIII-CLXXXIX further comprisesa linker L (e.g., wherein —O—R3 and/or —O—R4 as shown above is —O-L-R3and/or —O-L-R4), where L is a C1 to C10 alkyl, alkyl ether, polyether,polyethylene glycol, acetal, amide, succinyl, carbonyl, carbamide,carbamate, carbonate, ester (i.e., monoester, diester), or other linkeras is generally known in the art.

In one embodiment, a formulation of the invention (e.g., a formulatedmolecular compositions (FMC) or lipid nanoparticle (LNP) of theinvention) is a neutral lipid having any of formulae NLI-NLVII.

Examples of a steroid hormone include those comprising cholesterol,estrogen, testosterone, progesterone, glucocortisone, adrenaline,insulin, glucagon, cortisol, vitamin D, thyroid hormone, retinoic acid,and/or growth hormones.

In one embodiment, the invention features a composition comprising abiologically active molecule (e.g., a polynucleotide such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, other nucleic acid molecule or otherbiologically active molecule described herein), a cationic lipid, aneutral lipid, and a polyethyleneglycol conjugate, such as aPEG-diacylglycerol, PEG-diacylglycamide, PEG-cholesterol, or PEG-DMBconjugate. In another embodiment, the composition further comprisescholesterol or a cholesterol derivative. The compositions describedherein are generally referred to as formulated molecular compositions(FMC) or lipid nanoparticles (LNP). In some embodiments of theinvention, a formulated molecular composition (FMC) or lipidnanoparticle (LNP) composition further comprises cholesterol or acholesterol derivative.

Suitable cationic lipid include those cationic lipids which carry a netnegative charge at a selected pH, such as physiological pH. Particularlyuseful cationic lipids include those having a relatively small headgroup, such as a tertiary amine, quaternary amine or guanidine headgroup, and sterically hindered asymmetric lipid chains. In any of theembodiments described herein, the cationic lipid can be selected fromthose comprising Formulae CLI, CLII, CLIII, CLIV, CLV, CLVI, CLVII,CLVIII, CLIX, CLX, CLXI, CLXII, CLXIII, CLXIV, CLXV, CLXVI, CLXVI,CLXVII, CLXVIII, CLXIX, CLXX, CLXXI, CLXXII, CLXXIII, CLXXIV, CLXXV,CLXXVI, CLXXVII, CLXXVIII, CLXXIX, CLXXX, CLXXXI, CLXXXII, CLXXXIII,CLXXXIV, CLXXXV, CLXXXVI, CLXXXVII, CLXXXVIII, CLXXXIX, CLXXXX, CLXXXXI,CLXXXXII CLXXXX, CLXXXXI, CLXXXXII, CLXXXXIII, CLXXXXIV, CLXXXXV,CLXXXXVI; CLXXXXIII; CLXXXXIV; CLXXXXV; CLXXXXVI;N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-Dioleoyl-3-Dimethylammonium-propane (DODAP),1,2-Dioleoylcarbamyl-3-Dimethylammonium-propane (DOCDAP),1,2-Dilineoyl-3-Dimethylammonium-propane (DLINDAP),Dioleoyloxy-N-[2-sperminecarboxamido)ethyl}-N,N-dimethyl-1-propanaminiumtrifluoroacetate(DOSPA), Dioctadecylamidoglycyl spermine (DOGS), DC-Chol,1,2-Dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide(DMRIE),3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA), 2-[5′-(cholest-5-en-3β-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,12′-octadecadienoxy)propane (CpLinDMA),N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA),1,2-N,N′-Dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), and/or amixture thereof, as well as other cationic lipids sharing similarproperties. The above cationic lipids can include various differingsalts as are known in the art. Non-limiting examples of these cationiclipid structures are shown in FIGS. 7-11.

In some embodiments, the head group of the cationic lipid can beattached to the lipid chain via a cleavable or non-cleavable linker,such as a linker described herein or otherwise known in the art.Non-limiting examples of suitable linkers include those comprising a C1to C10 alkyl, alkyl ether, polyether, polyethylene glycol, acetal,amide, carbonyl, carbamide, carbamate, carbonate, ester (i.e.,monoester, diester), or succinyl.

Suitable neutral lipids include those comprising any of a variety ofneutral uncharged, zwitterionic or anionic lipids capable of producing astable complex. They are preferably neutral, although they canalternatively be positively or negatively charged. In any of theembodiments described herein, suitable neutral lipids include thoseselected from compounds having formulae NLI-NLVII,dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine(EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), -phosphatidylet-hanolamine (POPE) anddioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), cholesterol,as well as other neutral lipids described herein below, and/or a mixturethereof.

Suitable polyethyleneglycol-diacylglycerol orpolyethyleneglycol-diacylglycamide (PEG-DAG) conjugates include thosecomprising a dialkylglycerol or dialkylglycamide group having alkylchain length independently comprising from about C4 to about C40saturated or unsaturated carbon atoms. The dialkylglycerol ordialkylglycamide group can further comprise one or more substitutedalkyl groups. In any of the embodiments described herein, the PEGconjugate can be selected from PEG-dilaurylglycerol (C12),PEG-dimyristylglycerol (C14), PEG-dipalmitoylglycerol (C16),PEG-disterylglycerol (C18), PEG-dilaurylglycamide (C12),PEG-dimyristylglycamide (C14), PEG-dipalmitoylglycamide (C16), andPEG-disterylglycamide (C18), PEG-cholesterol(1-[8′-(Cholest-5-en-3β-oxy)carboxamido-3′,6′-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethyleneglycol), and PEG-DMB (3,4-Ditetradecoxylbenzyl-ω-methyl-poly(ethyleneglycol) ether).

In one embodiment, the invention features a composition comprising abiologically active molecule (e.g., a polynucleotide such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule)formulated as L051, L053, L054, L060, L061, L069, L073, L077, L080,L082, L083, L086, L097, L098, L099, L100, L101, L102, L103, L104, L105,L106, L107, L108, L109, L110, L111, L112, L113, L114, L115, L116, L117,L118, L121, L122, L123, L124, L130, L131, L132, L133, L134, L149, L155,L156, L162, L163, L164, L165, L166, L167, L174, L175, L176, L180, L181,and/or L182 herein (see Table IV).

Other suitable PEG conjugates include PEG-cholesterol or PEG-DMBconjugates. In one embodiment, PEG conjugates include PEGs attached tosaturated or unsaturated lipid chains such as oleyl, linoleyl andsimilar lipid chains.

In one embodiment, the invention features a composition comprising abiologically active molecule (e.g., a polynucleotide such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule), acationic lipid having any of Formulae CLI-CLXXXXVI, a neutral lipid, anda PEG-DAG (i.e., polyethyleneglycol-diacylglycerol orpolyethyleneglycol-diacylglycamide), PEG-cholesterol, or PEG-DMBconjugate. In another embodiment, the composition further comprisescholesterol or a cholesterol derivative. In another embodiment, thecomposition is formulated as L051, L053, L054, L060, L061, L069, L073,L077, L080, L082, L083, L086, L097, L098, L099, L100, L101, L102, L103,L104, L105, L106, L107, L108, L109, L110, L111, L112, L113, L114, L115,L116, L117, L118, L121, L122, L123, L124, L130, L131, L132, L133, L134,L149, L155, L156, L162, L163, L164, L165, L166, L167, L174, L175, L176,L180, L181, and/or L182 herein (see Table IV).

In one embodiment, the invention features a composition comprising abiologically active molecule (e.g., a polynucleotide such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule), acationic lipid comprising3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA), a neutral lipid comprising distearoylphosphatidylcholine(DSPC), a PEG-DAG comprising PEG-n-dimyristylglycerol (PEG-DMG), andcholesterol. In one embodiment, the molar ratio ofCLinDMA:DSPC:cholesterol:PEG-DMG are 48:40:10:2 respectively, thiscomposition is generally referred to herein as formulation L051.

In one embodiment, the invention features a composition comprising abiologically active molecule (e.g., a polynucleotide such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule), acationic lipid comprising N,N-Dimethyl-3,4-dioleyloxybenzylamine(DMOBA), a neutral lipid comprising distearoylphosphatidylcholine(DSPC), a PEG-DAG comprising PEG-n-dimyristylglycerol (PEG-DMG), andcholesterol. In one embodiment, the molar ratio ofDMOBA:DSPC:cholesterol:PEG-DMG are 30:20:48:2 respectively, thiscomposition is generally referred to herein as formulation L053.

In one embodiment, the invention features a composition comprising abiologically active molecule (e.g., a polynucleotide such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule), acationic lipid comprising N,N-Dimethyl-3,4-dioleyloxybenzylamine(DMOBA), a neutral lipid comprising distearoylphosphatidylcholine(DSPC)? a PEG-DAG comprising PEG-n-dimyristylglycerol (PEG-DMG), andcholesterol. In one embodiment, the molar ratio ofDMOBA:DSPC:cholesterol:PEG-DMG are 50:20:28:2 respectively, thiscomposition is generally referred to herein as formulation L054. Inanother embodiment, the composition further comprises a neutral lipid,such as dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine(EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and/or amixture thereof.

In one embodiment, the invention features a composition comprising abiologically active molecule (e.g., a polynucleotide such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule), acationic lipid comprising3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA), a cationic lipid comprisingN,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA), a neutral lipidcomprising distearoylphosphatidylcholine (DSPC), a PEG-DAG comprisingPEG-n-dimyristylglycerol (PEG-DMG), and cholesterol. In one embodiment,the molar ratio of CLinDMA:DMOBA:DSPC:cholesterol:PEG-DMG are25:25:20:28:2 respectively, this composition is generally referred toherein as formulation L03.

In one embodiment, the invention features a composition comprising abiologically active molecule (e.g., a polynucleotide such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule), acationic lipid comprising3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA), a neutral lipid comprising distearoylphosphatidylcholine(DSPC), a PEG comprising PEG-Cholesterol (PEG-Chol), and cholesterol. Inone embodiment, the molar ratio of CLinDMA:DSPC:cholesterol:PEG-Chol are48:40:10:2 respectively, this composition is generally referred toherein as formulation L069.

In one embodiment, the invention features a composition comprising abiologically active molecule (e.g., a polynucleotide such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule), acationic lipid comprising1,2-N,N′-Dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), a neutrallipid comprising distearoylphosphatidylcholine (DSPC), a PEG-DAGcomprising PEG-n-dimyristylglycerol (PEG-DMG), and cholesterol. In oneembodiment, the molar ratio of DOcarbDAP:DSPC:cholesterol:PEG-DMG are30:20:48:2 respectively, this composition is generally referred toherein as formulation T018.1.

In one embodiment, the invention features a composition comprising abiologically active molecule (e.g., a polynucleotide such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule), acationic lipid comprising N,N-dimethyl-2,3-dioleyloxy)propylamine(DODMA), a neutral lipid comprising distearoylphosphatidylcholine(DSPC), a PEG-DAG comprising PEG-n-dimyristylglycerol (PEG-DMG), andcholesterol. In one embodiment, the molar ratio ofDODMA:DSPC:cholesterol:PEG-DMG are 30:20:48:2 respectively, thiscomposition is generally referred to herein as formulation T019.1.

In one embodiment, the invention features a composition comprising abiologically active molecule (e.g., a polynucleotide such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule), and acationic lipid comprising a compound having any of Formula CLI, CLII,CLIII, CLIV, CLV, CLVI, CLVII, CLVIII, CLIX, CLX, CLXI, CLXII, CLXIII,CLXIV, CLXV, CLXVI, CLXVII, CLXVIII, CLXIX, CLXX, CLXXI, CLXXII,CLXXIII, CLXXIV, CLXXV, CLXXVI, CLXXVII, CLXXVIII, CLXXIX, CLXXX,CLXXXI, CLXXXII, CLXXXIII, CLXXXIV, CLXXXV, CLXXXVI, CLXXXVII,CLXXXVIII, CLXXXIX, CLXXXX, CLXXXXI, CLXXXXII CLXXXX, CLXXXXI, CLXXXXII,CLXXXXIII, CLXXXIV, CLXXXXV, CLXXXXVI, CLXXXXIII, CLXXXXIV, CLXXXXV, orCLXXXXVI. In another embodiment, the composition further comprises aneutral lipid, such as dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine(EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and/or amixture thereof. In another embodiment, the composition furthercomprises a PEG conjugate. In yet another embodiment, the compositionfurther comprises cholesterol or a cholesterol derivative.

In one embodiment, the invention features a composition comprising abiologically active molecule (e.g., a polynucleotide such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule), and acationic lipid comprising3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA). In another embodiment, the composition further comprises aneutral lipid, such as dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine(EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and/or amixture thereof. In another embodiment, the composition furthercomprises a PEG conjugate (i.e., polyethyleneglycol diacylglycerol(PEG-DAG), PEG-cholesterol, or PEG-DMB). In yet another embodiment, thecomposition further comprises cholesterol or a cholesterol derivative.

In one embodiment, the invention features a composition comprising abiologically active molecule (e.g., a polynucleotide such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule), and acationic lipid comprising N,N-Dimethyl-3,4-dioleyloxybenzylamine(DMOBA). In another embodiment, the composition further comprises aneutral lipid, such as dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine(EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and/or amixture thereof. In yet another embodiment, the composition furthercomprises the cationic lipid CLinDMA. In another embodiment, thecomposition further comprises a PEG conjugate. In yet anotherembodiment, the composition further comprises cholesterol or acholesterol derivative.

In one embodiment, a cationic lipid of the invention include thosecationic lipids which carry a net negative charge at a selected pH, suchas physiological pH. Particularly useful cationic lipids include thosehaving a relatively small head group, such as a tertiary amine,quaternary amine or guanidine head group, and sterically hinderedasymmetric lipid chains. In any of the embodiments described herein, thecationic lipid can be selected from those comprising Formulae CLI, CLII,CLIII, CLIV, CLV, CLVI, CLVII, CLVIII, CLIX, CLX, CLXI, CLXII, CLXIII,CLXIV, CLXV, CLXVI, CLXVI, CLXVII, CLXVIII, CLXIX, CLXX, CLXXI, CLXXII,CLXXIII, CLXXIV, CLXXV, CLXXVI, CLXXVII, CLXXVIII, CLXXIX, CLXXX,CLXXXI, CLXXXII, CLXXXIII, CLXXXIV, CLXXXV, CLXXXVI, CLXXXVII,CLXXXVIII, CLXXXIX, CLXXXX, CLXXXXI, CLXXXXII CLXXXX, CLXXXXI, CLXXXXII,CLXXXXIII, CLXXXXIV, CLXXXXV, CLXXXXVI (see U.S. Ser. No. 11/586,102);N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-Dioleoyl-3-Dimethylammonium-propane (DODAP),1,2-Dioleoylcarbamyl-3-Dimethylammonium-propane (DOCDAP),1,2-Dilineoyl-3-Dimethylammonium-propane (DLINDAP),Dioleoyloxy-N-[2-sperminecarboxamido)ethyl}-N,N-dimethyl-1-propanaminiumtrifluoroacetate(DOSPA), Dioctadecylamidoglycyl spermine (DOGS), DC-Chol,1,2-Dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide(DMRIE),3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA), 2-[5′-(cholest-5-en-3β-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,12′-octadecadienoxy)propane (CpLinDMA),N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA),1,2-N,N′-Dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), and/or amixture thereof, as well as other cationic lipids sharing similarproperties. The above cationic lipids can include various differingsalts as are known in the art. Non-limiting examples of these cationiclipid structures are shown in U.S. Ser. No. 11/586,102.

In some embodiments, the head group of the cationic lipid can beattached to the lipid chain via a cleavable or non-cleavable linker,such as a linker described herein or otherwise known in the art.Non-limiting examples of suitable linkers include those comprising a C1to C10 alkyl, alkyl ether, polyether, polyethylene glycol, acetal,amide, carbonyl, carbamide, carbamate, carbonate, ester (i.e.,monoester, diester), or succinyl.

In one embodiment, a neutral lipid of the invention includes thosecomprising any of a variety of neutral uncharged, zwitterionic oranionic lipids capable of producing a stable complex. They arepreferably neutral, although they can alternatively be positively ornegatively charged. In any of the embodiments described herein, suitableneutral lipids include those selected from compounds having formulaeNLI-NLVII, dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine(EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), -phosphatidylet-hanolamine (POPE) anddioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), cholesterol,as well as other neutral lipids described herein below, and/or a mixturethereof.

In one embodiment, the polyethyleneglycol-diacylglycerol orpolyethyleneglycol-diacylglycamide (PEG-DAG) conjugates of the inventioninclude those comprising a dialkylglycerol or dialkylglycamide grouphaving alkyl chain length independently comprising from about C4 toabout C40 saturated or unsaturated carbon atoms. The dialkylglycerol ordialkylglycamide group can further comprise one or more substitutedalkyl groups. In any of the embodiments described herein, the PEGconjugate can be selected from PEG-dilaurylglycerol (C12),PEG-dimyristylglycerol (C14), PEG-dipalmitoylglycerol (C16),PEG-disterylglycerol (C18), PEG-dilaurylglycamide (C12),PEG-dimyristylglycamide (C14), PEG-dipalmitoylglycamide (C16), andPEG-disterylglycamide (C18), PEG-cholesterol(1-[8′-(Cholest-5-en-3β-oxy)carboxamido-3′,6′-dioxaoctanyl]carbamoyl-comethyl-poly(ethylene glycol), and PEG-DMB(3,4-Ditetradecoxylbenzyl-ω-methyl-poly(ethylene glycol) ether).

In one embodiment, a formulation or vehicle of the invention comprises acomposition (e.g., one or more biologically active molecules and/or oneor more carrier molecules) formulated as L051, L053, L054, L060, L061,L069, L073, L077, L080, L082, L083, L086, L097, L098, L099, L100, L101,L102, L103, L104, L105, L106, L107, L108, L109, L110, L111, L112, L113,L114, L115, L116, L117, L118, L121, L122, L123, L124, L130, L131, L132,L133, L134, L149, L155, L156, L162, L163, L164, L165, L166, L167, L174,L175, L176, L180, L181, and/or L182 herein (see Table IV).

In one embodiment, a composition of the invention further comprises atargeting ligand for a specific cell of tissue type. Non-limitingexamples of such ligands include sugars and carbohydrates such asgalactose, galactosamine, and N-acetyl galactosamine; hormones such asestrogen, testosterone, progesterone, glucocortisone, adrenaline,insulin, glucagon, cortisol, vitamin D, thyroid hormone, retinoic acid,and growth hormones; growth factors such as VEGF, EGF, NGF, and PDGF;cholesterol; bile acids; neurotransmitters such as GABA, Glutamate,acetylcholine; NOGO; inostitol triphosphate; diacylglycerol;epinephrine; norepinephrine; Nitric Oxide, peptides, vitamins such asfolate and pyridoxine, drugs, antibodies and any other molecule that caninteract with a receptor in vivo or in vitro. The ligand can be attachedto any component of a formulated siNA composition of invention (e.g.,cationic lipid component, neutral lipid component, PEG-DAG component, orsiNA component etc.) using a linker molecule, such as an amide, amido,carbonyl, ester, peptide, disulphide, silane, nucleoside, abasicnucleoside, polyether, polyamine, polyamide, peptide, carbohydrate,lipid, polyhydrocarbon, phosphate ester, phosphoramidate, thiophosphate,alkylphosphate, or photolabile linker. In one embodiment, the linker isa biodegradable linker.

In one embodiment, the invention features a composition comprising asiNA molecule and/or a carrier molecule, a cationic lipid having any ofFormulae CLI-CLXXXXVI, a neutral lipid, and apolyethyleneglycol-diacylglycerol or polyethyleneglycol-diacylglycamide(PEG-DAG) conjugate (i.e., polyethyleneglycol diacylglycerol (PEG-DAG),PEG-cholesterol, or PEG-DMB). These compositions are generally referredto herein as formulated siNA compositions of LNP compositions. Inanother embodiment, a formulated siNA composition of the inventionfurther comprises cholesterol or a cholesterol derivative.

In one embodiment, the siNA component of a formulated siNA compositionof the invention is chemically modified so as not to stimulate aninterferon response in a mammalian cell, subject, or organism. Such siNAmolecules can be said to have improved toxicologic profiles, such ashaving attenuated or no immunostimulatory properties, having attenuatedor no off-target effect, or otherwise as described herein (see forexample PCT/US06/032168).

In one embodiment, the invention features a composition comprising amiRNA molecule and or a carrier molecule, a cationic lipid having any ofFormulae CLI-CLXXXXVI, a neutral lipid, and apolyethyleneglycol-diacylglycerol or polyethyleneglycol-diacylglycamide(PEG-DAG) conjugate (i.e., polyethyleneglycol diacylglycerol (PEG-DAG),PEG-cholesterol, or PEG-DMB). These compositions are generally referredto herein as formulated miRNA compositions. In another embodiment, aformulated miRNA composition of the invention further comprisescholesterol or a cholesterol derivative.

In one embodiment, the miRNA component of a formulated miRNA compositionof the invention is chemically modified so as not to stimulate aninterferon response in a mammalian cell, subject, or organism. SuchmiRNA molecules can be said to have improved toxicologic profiles, suchas having attenuated or no immunostimulatory properties, havingattenuated or no off-target effect, or otherwise as described herein.

In one embodiment, the invention features a composition comprising aRNAi inhibitor molecule and/or a carrier molecule, a cationic lipidhaving any of Formulae CLI-CLXXXXVI, a neutral lipid, and apolyethyleneglycol-diacylglycerol or polyethyleneglycol-diacylglycamide(PEG-DAG) conjugate (i.e., polyethyleneglycol diacylglycerol (PEG-DAG),PEG-cholesterol, or PEG-DMB). These compositions are generally referredto herein as formulated RNAi inhibitor compositions. In anotherembodiment, a formulated RNAi inhibitor composition of the inventionfurther comprises cholesterol or a cholesterol derivative.

In one embodiment, the RNAi inhibitor component of a formulated RNAiinhibitor composition of the invention is chemically modified so as notto stimulate an interferon response in a mammalian cell, subject, ororganism. Such RNAi inhibitor molecules can be said to have improvedtoxicologic profiles, such as having attenuated or no immunostimulatoryproperties, having attenuated or no off-target effect, or otherwise asdescribed herein

In one embodiment, the invention features a composition comprising: (a)a cationic lipid having any of Formulae CLI-CLXXXXVI; (b) a neutrallipid; (c) a polyethyleneglycol-diacylglycerol (PEG-DAG) conjugate(i.e., polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, orPEG-DMB); and (d) a carrier molecule and/or a short interfering nucleicacid (siNA) molecule that mediates RNA interference (RNAi) against RNAof a target gene, wherein each strand of said siNA molecule is about 18to about 28 nucleotides in length; and one strand of said siNA moleculecomprises nucleotide sequence having sufficient complementarity to thetarget gene RNA for the siNA molecule to mediate RNA interferenceagainst the target gene RNA. In one embodiment, the target RNA comprisesRNA sequence referred to by Genbank Accession numbers in InternationalPCT Publication No. WO 03/74654, serial No. PCT/US03/05028, and U.S.patent application Ser. No. 10/923,536 both incorporated by referenceherein. In another embodiment, the composition further comprisescholesterol or a cholesterol derivative.

In one embodiment, the invention features a composition comprising: (a)a cationic lipid having any of Formulae CLI-CLXXXXVI; (b) a neutrallipid; (c) a polyethyleneglycol-diacylglycerol (PEG-DAG) conjugate(i.e., polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, orPEG-DMB); and (d) a carrier molecule and/or a miRNA molecule thatmediates RNA interference (RNAi) against RNA of a target gene, whereineach strand of said miRNA molecule is about 18 to about 40 nucleotidesin length; and one strand of said miRNA molecule comprises nucleotidesequence having sufficient complementarity to the target gene RNA forthe miRNA molecule to mediate RNA interference against the target geneRNA. In one embodiment, the target RNA comprises RNA sequence referredto by Genbank Accession numbers in International PCT Publication No. WO03/74654, serial No. PCT/US03/05028, and U.S. patent application Ser.No. 10/923,536 both incorporated by reference herein. In anotherembodiment, the composition further comprises cholesterol or acholesterol derivative.

In one embodiment, the invention features a composition comprising: (a)a cationic lipid having any of Formulae CLI-CLXXXXVI; (b) a neutrallipid; (c) a polyethyleneglycol-diacylglycerol (PEG-DAG) conjugate(i.e., polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, orPEG-DMB); and (d) a carrier molecule and/or a RNAi inhibitor moleculethat modulates RNA interference (RNAi) activity of a miRNA or siRNAtarget, wherein said RNAi inhibitor molecule is about 15 to about 40nucleotides in length; and said RNAi inhibitor molecule comprisesnucleotide sequence having sufficient complementarity to the targetsiRNA or miRNA for the RNAi inhibitor molecule to modulate the RNAiactivity of the target siRNA or miRNA. In one embodiment, the miRNA orsiRNA target comprises RNA sequence comprising a portion of RNA sequencereferred to by Genbank Accession numbers in International PCTPublication No. WO 03/74654, serial No. PCT/US03/05028, and U.S. patentapplication Ser. No. 10/923,536 both incorporated by reference herein.In another embodiment, the composition further comprises cholesterol ora cholesterol derivative.

In one embodiment, the invention features a composition comprising: (a)a cationic lipid having any of Formulae CLI-CLXXXXVI; (b) a neutrallipid; (c) a polyethyleneglycol-diacylglycerol (PEG-DAG) conjugate(i.e., polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, orPEG-DMB); and (d) a carrier molecule and/or a short interfering nucleicacid (siNA) molecule that mediates RNA interference (RNAi) against aHepatitis Virus RNA, wherein each strand of said siNA molecule is about18 to about 28 nucleotides in length; and one strand of said siNAmolecule comprises nucleotide sequence having sufficient complementarityto the Hepatitis Virus RNA for the siNA molecule to mediate RNAinterference against the Hepatitis Virus RNA. In one embodiment, theHepatitis Virus RNA is Hepatitis B Virus (HBV). In one embodiment, theHepatitis Virus RNA is Hepatitis C Virus (HCV). In one embodiment, thesiNA comprises sequences described in U.S. Patent Application No.60/401,104, Ser. Nos. 10/667,271, and 10/942,560, which are incorporatedby reference in their entireties herein. In another embodiment, thecomposition further comprises cholesterol or a cholesterol derivative.

In one embodiment, the invention features a composition comprising: (a)a cationic lipid having any of Formulae CLI-CLXXXXVI; (b) a neutrallipid; (c) a polyethyleneglycol conjugate (i.e., polyethyleneglycoldiacylglycerol (PEG-DAG), PEG-cholesterol, or PEG-DMB); and (d) acarrier molecule and/or a short interfering nucleic acid (siNA) moleculethat mediates RNA interference (RNAi) against Protein TyrosinePhosphatase 1B (PTP1B) RNA, wherein each strand of said siNA molecule isabout 18 to about 28 nucleotides in length; and one strand of said siNAmolecule comprises nucleotide sequence having sufficient complementarityto the PTP1B RNA for the siNA molecule to mediate RNA interferenceagainst the PTP1B RNA. In one embodiment, the siNA comprises sequencesdescribed in U.S. Patent Application Publication Nos. 20040019001 and200500704978, which are incorporated by reference in their entiretiesherein. In another embodiment, the composition further comprisescholesterol or a cholesterol derivative.

In one embodiment, the invention features a composition comprising: (a)a cationic lipid having any of Formulae CLI-CLXXXXVI; (b) a neutrallipid; (c) a polyethyleneglycol conjugate (i.e., polyethyleneglycoldiacylglycerol (PEG-DAG), PEG-cholesterol, or PEG-DMB); and (d) acarrier molecule and/or a short interfering nucleic acid (siNA) moleculethat mediates RNA interference (RNAi) against Transforming Growth Factorbeta (TGF-beta) and/or Transforming Growth Factor beta Receptor(TGF-betaR) RNA, wherein each strand of said siNA molecule is about 18to about 28 nucleotides in length; and one strand of said siNA moleculecomprises nucleotide sequence having sufficient complementarity to theTGF-beta and/or TGF-betaR RNA for the siNA molecule to mediate RNAinterference against the TGF-beta and/or TGF-betaR RNA. In oneembodiment, the siNA comprises sequences described in U.S. Ser. No.11/054,047, which is incorporated by reference in their entiretiesherein. In another embodiment, the composition further comprisescholesterol or a cholesterol derivative.

In one embodiment, the invention features a composition comprising: (a)a cationic lipid having any of Formulae CLI-CLXXXXVI; (b) a neutrallipid; (c) a polyethyleneglycol conjugate (i.e., polyethyleneglycoldiacylglycerol (PEG-DAG), PEG-cholesterol, or PEG-DMB); and (d) acarrier molecule and/or a short interfering nucleic acid (siNA) moleculethat mediates RNA interference (RNAi) against cholesteryl ester transferprotein (CETP) RNA, wherein each strand of said siNA molecule is about18 to about 28 nucleotides in length; and one strand of said siNAmolecule comprises nucleotide sequence having sufficient complementarityto the CETP RNA for the siNA molecule to mediate RNA interferenceagainst the CETP RNA. In one embodiment, the siNA comprises sequencesdescribed in U.S. Ser. No. 10/921,554, which is incorporated byreference in its entirety herein. In another embodiment, the compositionfurther comprises cholesterol or a cholesterol derivative.

In one embodiment, the invention features a composition comprising: (a)a cationic lipid having any of Formulae CLI-CLXXXXVI; (b) a neutrallipid; (c) a polyethyleneglycol conjugate (i.e., polyethyleneglycoldiacylglycerol (PEG-DAG), PEG-cholesterol, or PEG-DMB); and (d) acarrier molecule and/or a short interfering nucleic acid (siNA) moleculethat mediates RNA interference (RNAi) against Gastric Inhibitory Peptide(GIP) RNA, wherein each strand of said siNA molecule is about 18 toabout 28 nucleotides in length; and one strand of said siNA moleculecomprises nucleotide sequence having sufficient complementarity to theGIP RNA for the siNA molecule to mediate RNA interference against theGIP RNA. In one embodiment, the siNA comprises sequences described inU.S. Ser. No. 10/916,030, which is incorporated by reference in itsentirety herein. In another embodiment, the composition furthercomprises cholesterol or a cholesterol derivative.

In one embodiment, the invention features a composition comprising: (a)a cationic lipid having any of Formulae CLI-CLXXXXVI; (b) a neutrallipid; (c) a polyethyleneglycol conjugate (i.e., polyethyleneglycoldiacylglycerol (PEG-DAG), PEG-cholesterol, or PEG-DMB); and (d) acarrier molecule and/or a short interfering nucleic acid (siNA) moleculethat mediates RNA interference (RNAi) against Stearoyl-CoA Desaturase(SCD) RNA, wherein each strand of said siNA molecule is about 18 toabout 28 nucleotides in length; and one strand of said siNA moleculecomprises nucleotide sequence having sufficient complementarity to theSCD RNA for the siNA molecule to mediate RNA interference against theSCD RNA. In one embodiment, the siNA comprises sequences described inU.S. Ser. No. 10/923,451, which is incorporated by reference in itsentirety herein. In another embodiment, the composition furthercomprises cholesterol or a cholesterol derivative.

In one embodiment, the invention features a composition comprising: (a)a cationic lipid having any of Formulae CLI-CLXXXXVI; (b) a neutrallipid; (c) a polyethyleneglycol-diacylglycerol conjugate (i.e.,polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, orPEG-DMB); and (d) a carrier molecule and/or a short interfering nucleicacid (siNA) molecule that mediates RNA interference (RNAi) againstAcetyl-CoA carboxylase (ACACB) RNA, wherein each strand of said siNAmolecule is about 18 to about 28 nucleotides in length; and one strandof said siNA molecule comprises nucleotide sequence having sufficientcomplementarity to the ACACB RNA for the siNA molecule to mediate RNAinterference against the ACACB RNA. In one embodiment, the siNAcomprises sequences described in U.S. Ser. No. 10/888,226, which isincorporated by reference in its entirety herein. In another embodiment,the composition further comprises cholesterol or a cholesterolderivative.

In one embodiment, the invention features a composition comprising: (a)a cationic lipid having any of Formulae CLI-CLXXXXVI; (b) a neutrallipid; (c) a polyethyleneglycol conjugate (i.e., polyethyleneglycoldiacylglycerol (PEG-DAG), PEG-cholesterol, or PEG-DMB); and (d) acarrier molecule and/or a short interfering nucleic acid (siNA) moleculethat mediates RNA interference (RNAi) against apolipoprotein RNA (e.g.,apo AI, apo A-IV, apo B, apo C-III, and/or apo E RNA), wherein eachstrand of said siNA molecule is about 18 to about 28 nucleotides inlength; and one strand of said siNA molecule comprises nucleotidesequence having sufficient complementarity to the apolipoprotein RNA forthe siNA molecule to mediate RNA interference against the apolipoproteinRNA. In one embodiment, the siNA comprises sequences described in U.S.Ser. No. 11/054,047, which is incorporated by reference in theirentireties herein. In another embodiment, the composition furthercomprises cholesterol or a cholesterol derivative.

In one embodiment, the invention features a composition comprising: (a)a cationic lipid having any of Formulae CLI-CLXXXXVI; (b) a neutrallipid; (c) a polyethyleneglycol conjugate (i.e., polyethyleneglycoldiacylglycerol (PEG-DAG), PEG-cholesterol, or PEG-DMB); and (d) acarrier molecule and/or a short interfering nucleic acid (siNA) moleculethat mediates RNA interference (RNAi) against VEGF and/or VEGF-receptorRNA (e.g., VEGF, VEGFR1, VEGFR2 and/or VEGFR3RNA), wherein each strandof said siNA molecule is about 18 to about 28 nucleotides in length; andone strand of said siNA molecule comprises nucleotide sequence havingsufficient complementarity to the VEGF and/or VEGF-receptor RNA for thesiNA molecule to mediate RNA interference against the VEGF and/orVEGF-receptor RNA. In one embodiment, the siNA comprises sequencesdescribed in U.S. Ser. No. 10/962,898, which is incorporated byreference in their entireties herein. In another embodiment, thecomposition further comprises cholesterol or a cholesterol derivative.

In one embodiment, the invention features a composition comprising: (a)a cationic lipid having any of Formulae CLI-CLXXXXVI; (b) a neutrallipid; (c) a polyethyleneglycol conjugate (i.e., polyethyleneglycoldiacylglycerol (PEG-DAG), PEG-cholesterol, or PEG-DMB); and (d) acarrier molecule and/or a short interfering nucleic acid (siNA) moleculethat mediates RNA interference (RNAi) against IL4-receptor RNA, whereineach strand of said siNA molecule is about 18 to about 28 nucleotides inlength; and one strand of said siNA molecule comprises nucleotidesequence having sufficient complementarity to the IN-receptor RNA forthe siNA molecule to mediate RNA interference against the IL4-receptorRNA. In one embodiment, the siNA comprises sequences described in U.S.Ser. No. 11/001,347, which is incorporated by reference in theirentireties herein. In another embodiment, the composition furthercomprises cholesterol or a cholesterol derivative.

In one embodiment, the invention features a composition comprising: (a)a cationic lipid having any of Formulae CLI-CLXXXXVI; (b) a neutrallipid; (c) a polyethyleneglycol conjugate (i.e., polyethyleneglycoldiacylglycerol (PEG-DAG), PEG-cholesterol, or PEG-DMB); and (d) acarrier molecule and/or a short interfering nucleic acid (siNA) moleculethat mediates RNA interference (RNAi) against Hairless RNA, wherein eachstrand of said siNA molecule is about 18 to about 28 nucleotides inlength; and one strand of said siNA molecule comprises nucleotidesequence having sufficient complementarity to the Hairless RNA for thesiNA molecule to mediate RNA interference against the Hairless RNA. Inone embodiment, the siNA comprises sequences described in U.S. Ser. No.10/919,964, which is incorporated by reference in their entiretiesherein. In another embodiment, the composition further comprisescholesterol or a cholesterol derivative.

In one embodiment, the invention features a composition comprising: (a)a cationic lipid having any of Formulae CLI-CLXXXXVI; (b) a neutrallipid; (c) a polyethyleneglycol conjugate (i.e., polyethyleneglycoldiacylglycerol (PEG-DAG), PEG-cholesterol, or PEG-DMB); and (d) acarrier molecule and/or a short interfering nucleic acid (siNA) moleculethat mediates RNA interference (RNAi) against a target RNA, wherein eachstrand of said siNA molecule is about 18 to about 28 nucleotides inlength; and one strand of said siNA molecule comprises nucleotidesequence having sufficient complementarity to the target RNA for thesiNA molecule to mediate RNA interference against the target RNA. In oneembodiment, the target RNA comprises RNA sequence referred to by GenbankAccession numbers in International PCT Publication No. WO 03/74654,serial No. PCT/US03/05028, and U.S. patent application Ser. No.10/923,536 both incorporated by reference herein. In another embodiment,the composition further comprises cholesterol or a cholesterolderivative.

In one embodiment, the cationic lipid component (e.g., a compound havingany of Formulae CLI-CLXXXXVI or as otherwise described herein) of acomposition of invention comprises from about 2% to about 60%, fromabout 5% to about 45%, from about 5% to about 15%, or from about 40% toabout 50% of the total lipid present in the formulation.

In one embodiment, the neutral lipid component of a composition of theinvention comprises from about 5% to about 90%, or from about 20% toabout 85% of the total lipid present in the formulation.

In one embodiment, the PEG conjugate (i.e., PEG-DAG, PEG-cholesterol,PEG-DMB) of a composition of the invention comprises from about 1% toabout 20%, or from about 4% to about 15% of the total lipid present inthe formulation.

In one embodiment, the cholesterol component of a composition of theinvention comprises from about 10% to about 60%, or from about 20% toabout 45% of the total lipid present in the formulation.

In one embodiment, a formulated siNA composition of the inventioncomprises a cationic lipid component comprising from about 30 to about50% of the total lipid present in the formulation, a neutral lipidcomprising from about 30 to about 50% of the total lipid present in theformulation, and a PEG conjugate (i.e., PEG-DAG, PEG-cholesterol,PEG-DMB) comprising about 0 to about 10% of the total lipid present inthe formulation.

In one embodiment, a formulated molecular composition of the inventioncomprises a biologically active molecule (e.g., a polynucleotide such asa siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme,2-5A, triplex forming oligonucleotide, or other nucleic acid molecule),a compound having any of Formulae CLI-CLXXXXVI, DSPC, and a PEGconjugate (i.e., PEG-DAG, PEG-cholesterol, PEG-DMB). In one embodiment,the PEG conjugate is PEG-dilaurylglycerol (C12), PEG-dimyristylglycerol(C14), PEG-dipalmitoylglycerol (C16), or PEG-disterylglycerol (C18). Inanother embodiment, the PEG conjugate is PEG-dilaurylglycamide (C12),PEG-dimyristylglycamide (C14), PEG-dipalmitoylglycamide (C16), orPEG-disterylglycamide (C18). In another embodiment, the PEG conjugate isPEG-cholesterol or PEG-DMB. In another embodiment, the formulatedmolecular composition further comprises cholesterol or a cholesterolderivative.

In one embodiment, a formulated molecular composition of the inventioncomprises a biologically active molecule (e.g., a polynucleotide such asa siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme,2-5A, triplex forming oligonucleotide, or other nucleic acid molecule),a compound having Formula CLI, DSPC, and a PEG conjugate. In oneembodiment, the PEG conjugate is PEG-dilaurylglycerol (C12),PEG-dimyristylglycerol (C14), PEG-dipalmitoylglycerol (C16), orPEG-disterylglycerol (C18). In another embodiment, the PEG conjugate isPEG-dilaurylglycamide (C12), PEG-dimyristylglycamide (C14),PEG-dipalmitoylglycamide (C16), or PEG-disterylglycamide (C18). Inanother embodiment, the PEG conjugate is PEG-cholesterol or PEG-DMB. Inanother embodiment, the formulated molecular composition furthercomprises cholesterol or a cholesterol derivative.

In one embodiment, a formulated molecular composition of the inventioncomprises a biologically active molecule (e.g., a polynucleotide such asa siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme,2-5A, triplex forming oligonucleotide, or other nucleic acid molecule),a compound having Formula CLV, DSPC, and a PEG conjugate. In oneembodiment, the PEG conjugate is PEG-dilaurylglycerol (C12),PEG-dimyristylglycerol (C14), PEG-dipalmitoylglycerol (C16), orPEG-disterylglycerol (C18). In another embodiment, the PEG conjugate isPEG-dilaurylglycamide (C12), PEG-dimyristylglycamide (C14),PEG-dipalmitoylglycamide (C16), or PEG-disterylglycamide (C18). Inanother embodiment, the PEG conjugate is PEG-cholesterol or PEG-DMB. Inanother embodiment, the formulated molecular composition furthercomprises cholesterol or a cholesterol derivative.

In one embodiment, a composition of the invention (e.g., a formulatedmolecular composition) further comprises a targeting ligand for aspecific cell of tissue type. Non-limiting examples of such ligandsinclude sugars and carbohydrates such as galactose, galactosamine, andN-acetyl galactosamine; hormones such as estrogen, testosterone,progesterone, glucocortisone, adrenaline, insulin, glucagon, cortisol,vitamin D, thyroid hormone, retinoic acid, and growth hormones; growthfactors such as VEGF, EGF, NGF, and PDGF; cholesterol; bile acids;neurotransmitters such as GABA, Glutamate, acetylcholine; NOGO;inostitol triphosphate; diacylglycerol; epinephrine; norepinephrine;Nitric Oxide, peptides, vitamins such as folate and pyridoxine, drugs,antibodies and any other molecule that can interact with a receptor invivo or in vitro. The ligand can be attached to any component of aformulated siNA composition of invention (e.g., cationic lipidcomponent, neutral lipid component, PEG-DAG component, or siNA componentetc.) using a linker molecule, such as an amide, amido, carbonyl, ester,peptide, disulphide, silane, nucleoside, abasic nucleoside, polyether,polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon,phosphate ester, phosphoramidate, thiophosphate, alkylphosphate, orphotolabile linker. In one embodiment, the linker is a biodegradablelinker.

In one embodiment, the PEG conjugate of the invention, such as aPEG-DAG, PEG-cholesterol, PEG-DMB, comprises a 200 to 10,000 atom PEGmolecule.

In one embodiment, the compositions of the present invention, e.g., aformulated molecular composition, comprise adiacylglycerol-polyethyleneglycol conjugate, i.e., a DAG-PEG conjugate.The term “diacylglycerol” refers to a compound having 2-fatty acylchains, R1 and R2, both of which have independently between 2 and 30carbons bonded to the I— and 2-position of glycerol by ester linkages.The acyl groups can be saturated or have varying degrees ofunsaturation. Diacylglycerols have the following general Formula VIII:

wherein R1 and R2 are each an alkyl, substituted alkyl, aryl,substituted aryl, lipid, or a ligand. In one embodiment, R1 and R2 areeach independently a C2 to C30 alkyl group. In one embodiment, theDAG-PEG conjugate is a dilaurylglycerol (C12)-PEG conjugate, adimyristylglycerol (C14)-PEG conjugate, a dipalmitoylglycerol (C16)-PEGconjugate, a disterylglycerol (C18)-PEG conjugate, PEG-dilaurylglycamide(C12), PEG-dimyristylglycamide (C14), PEG-dipalmitoylglycamide (C16), orPEG-disterylglycamide (C18). Those of skill in the art will readilyappreciate that other diacylglycerols can be used in the DAG-PEGconjugates of the present invention.

In one embodiment, the compositions of the present invention, e.g., aformulated molecular composition, comprise apolyethyleneglycol-cholesterol conjugate, i.e., a PEG-chol conjugate.The PEG-chol conjugate can comprise a 200 to 10,000 atom PEG moleculelinked to cholesterol or a cholesterol derivative. An exemplary PEG-choland the synthesis thereof is shown in FIG. 30.

In one embodiment, the compositions of the present invention, e.g., aformulated molecular composition, comprise a polyethyleneglycol-DMBconjugate. The term “DMB” refers to the compound3,4-Ditetradecoxylbenzyl-β-methyl-poly(ethylene glycol) ether. ThePEG-DMB conjugate can comprise a 200 to 10,000 atom PEG molecule linkedto DMB. An exemplary PEG-DMB and the synthesis thereof is shown in FIG.30A.

In one embodiment, the compositions of the present invention, e.g., aformulated molecular composition, comprise a PEG-lipid such as apolyethyleneglycol-DMG (PEG-DMG) conjugate. The term “PEG-DMG” can referto the compoundI-[8′-(1,2-Dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethyleneglycol). The PEG-DMG conjugate can comprise a 200 to 10,000 atom PEGmolecule linked to DMG moiety. In one embodiment, PEG is apolydispersion represented by the formula PEG_(n), where n=about 33 to67 for a 1500 Da to 3000 Da PEG, average=45 for 2 KPEG/PEG2000. Anexemplary PEG-DMG and the synthesis thereof is shown in FIG. 30B.

The term “ligand” refers to any compound or molecule, such as a drug,peptide, hormone, or neurotransmitter that is capable of interactingwith another compound, such as a receptor, either directly orindirectly. The receptor that interacts with a ligand can be present onthe surface of a cell or can alternately be an intercellular receptor.Interaction of the ligand with the receptor can result in a biochemicalreaction, or can simply be a physical interaction or association.Non-limiting examples of ligands include sugars and carbohydrates suchas galactose, galactosamine, and N-acetyl galactosamine; hormones suchas estrogen, testosterone, progesterone, glucocortisone, adrenaline,insulin, glucagon, cortisol, vitamin D, thyroid hormone, retinoic acid,and growth hormones; growth factors such as VEGF, EGF, NGF, and PDGF;cholesterol; bile acids; neurotransmitters such as GABA, Glutamate,acetylcholine; NOGO; inostitol triphosphate; diacylglycerol;epinephrine; norepinephrine; Nitric Oxide, peptides, vitamins such asfolate and pyridoxine, drugs, antibodies and any other molecule that caninteract with a receptor in vivo or in vitro. The ligand can be attachedto a compound of the invention using a linker molecule, such as anamide, amido, carbonyl, ester, peptide, disulphide, silane, nucleoside,abasic nucleoside, polyether, polyamine, polyamide, peptide,carbohydrate, lipid, polyhydrocarbon, phosphate ester, phosphoramidate,thiophosphate, alkylphosphate, or photolabile linker. In one embodiment,the linker is a biodegradable linker.

The term “degradable linker” as used herein, refers to linker moietiesthat are capable of cleavage under various conditions. Conditionssuitable for cleavage can include but are not limited to pH, UVirradiation, enzymatic activity, temperature, hydrolysis, elimination,and substitution reactions, and thermodynamic properties of the linkage.

The term “photolabile linker” as used herein, refers to linker moietiesas are known in the art that are selectively cleaved under particular UVwavelengths. Compounds of the invention containing photolabile linkerscan be used to deliver compounds to a target cell or tissue of interest,and can be subsequently released in the presence of a UV source.

The term “lipid” as used herein, refers to any lipophilic compound.Non-limiting examples of lipid compounds include fatty acids and theirderivatives, including straight chain, branched chain, saturated andunsaturated fatty acids, carotenoids, terpenes, bile acids, andsteroids, including cholesterol and derivatives or analogs thereof.

The term “PEG-lipid” as used herein, refers to any lipophilic compoundthat is covalently attached to a PEG moiety. Non-limiting examples ofPEG-lipids of the invention include PEG-ceramide conjugates, PEG-DAGconjugates and PEG-cholesterol conjugates as described herein or asotherwise known in the art. In one embodiment, PEG is a polydispersionrepresented by the formula PEG_(n), where n=about 33 to 67 for a 1500 Dato 3000 Da PEG, average=45 for 2 KPEG/PEG2000. An exemplary PEG-DAG andthe synthesis thereof is shown in FIG. 30B.

The term “formulation” as used herein, refers to any formulatedcomposition including one or more biologically active molecules, one ormore carrier molecules, or both biologically active molecules andcarrier molecules, along with any other components that allowintracellular delivery of the biologically active molecules and/orcarrier molecules. In one embodiment, the formulation is a lipidnanoparticle formulation as described herein (see Table IV) or asotherwise known in the art.

Suitable formulations for use in the present invention, and methods ofmaking and using such formulations are disclosed, for example in U.S.Patent Application Publication No. 20060240554 and U.S. Ser. No.11/586,102, filed Oct. 24, 2006; International PCT Publication No.WO2007012191, and U.S. Patent Application Publication Nos. 2006083780,2006051405, US2005175682, US2004142025, US2003077829, US2006240093, allof which are incorporated by reference herein in their entirety.

The invention additionally provides methods for determining whether aformulation or composition will be effective for delivery of abiologically active molecule into a biological system. In oneembodiment, the method for determining whether a formulation orcomposition will be effective for delivery of a biologically activemolecule into a biological system comprises (1) measuring the serumstability of the formulation or composition and (2) measuring the pHdependent phase transition of the formulation or composition, wherein adetermination that the formulation or composition is stable in serum anda determination that the formulation or composition undergoes a phasetransition at about pH 4 to about 7, e.g., from 5.5 to 6.5, indicatesthat the formulation or composition will be effective for delivery of abiologically active molecule into a biological system. In anotherembodiment, the method further comprises measuring the transfectionefficiency of the formulation or composition in a cell in vitro.

The serum stability of the formulation or composition can be measuredusing any assay that measures the stability of the formulation orcomposition in serum, including the assays described herein andotherwise known in the art. One exemplary assay that can be used tomeasure the serum stability is an assay that measures the relativeturbidity of the composition in serum over time. For example, therelative turbidity of a formulation or composition can be determined bymeasuring the absorbance of the formulation or composition in thepresence or absence of serum (i.e., 50%) at several time points over a24 hour period using a spectrophotometer. The formulation or compositionis stable in serum if the relative turbidity, as measured by absorbance,remains constant at around 1.0 over time.

The pH dependent phase transition of the formulation or composition canbe measured using any assay that measures the phase transition of theformulation or composition at about pH 5.5-6.5, including the assaysdescribed herein and otherwise known in the art. One exemplary assaythat can be used to measure the pH dependent phase transition is anassay that measures the relative turbidity of the composition atdifferent pH over time. For example, the relative turbidity of aformulation or composition can be determined by measuring the absorbanceover time of the formulation or composition in buffer having a range ofdifferent pH values. The formulation or composition undergoes pHdependent phase transition if the relative turbidity, as measured byabsorbance, decreases when the pH drops below 7.0.

In addition, the efficiency of the formulation or composition thatundergoes a rapid pH-dependent phase transition as a delivery agent canbe determined by measuring the transfection efficiency of theformulation or composition. Methods for performing transfection assaysare described herein and otherwise known in the art.

In one embodiment, the particles made by the methods of this inventionhave a size of about 50 to about 600 nm. The particles can be formed byeither a detergent dialysis method or by a modification of areverse-phase method which utilizes organic solvents to provide a singlephase during mixing of the components. Without intending to be bound byany particular mechanism of formation, a molecule (e.g., a biologicallyactive molecule such as a polynucleotide) is contacted with a detergentsolution of cationic lipids to form a coated molecular complex. Thesecoated molecules can aggregate and precipitate. However, the presence ofa detergent reduces this aggregation and allows the coated molecules toreact with excess lipids (typically, noncationic lipids) to formparticles in which the molecule of interest is encapsulated in a lipidbilayer. The methods described below for the formation of formulation orcomposition s using organic solvents follow a similar scheme.

In one embodiment, the particles are formed using detergent dialysis.Thus, the present invention provides a method for the preparation ofserum-stable formulation or composition s, including those that undergopH dependent phase transition, comprising: (a) combining a molecule(e.g., a biologically active molecule such as a polynucleotide,including siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleic acidmolecules) with cationic lipids in a detergent solution to form a coatedmolecule-lipid complex; (b) contacting noncationic lipids with thecoated molecule-lipid complex to form a detergent solution comprising asiNA-lipid complex and noncationic lipids; and (c) dialyzing thedetergent solution of step (b) to provide a solution of serum-stablemolecule-lipid particles, wherein the molecule is encapsulated in alipid bilayer and the particles are serum-stable and have a size of fromabout 50 to about 600 nm.

In one embodiment, an initial solution of coated molecule-lipid (e.g.,polynucleotide-lipid) complexes is formed, for example, by combining themolecule with the cationic lipids in a detergent solution.

In these embodiments, the detergent solution is preferably an aqueoussolution of a neutral detergent having a critical micelle concentrationof 15-300 mM, more preferably 20-50 mM. Examples of suitable detergentsinclude, for example,N,N′-((octanoylimino)-bis-(trimethylene))-bis-(D-gluconamide) (BIGCHAP);BRIJ 35; Deoxy-BIGCHAP; dodecylpoly(ethylene glycol) ether; Tween 20;Tween 40; Tween 60; Tween 80; Tween 85; Mega 8; Mega 9; Zwittergent®3-08; Zwittergent® 3-10; Triton X-405; hexyl-, heptyl-, octyl- andnonyl-beta-D-glucopyranoside; and heptylthioglucopyranoside; with octylβ-D-glucopyranoside and Tween-20 being the most preferred. Theconcentration of detergent in the detergent solution is typically about100 mM to about 2 M, preferably from about 200 mM to about 1.5 M.

In one embodiment, the cationic lipids and the molecule of interest(e.g., a biologically active molecule such as a polynucleotide,including siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleic acidmolecules) will typically be combined to produce a charge ratio (+/−) ofabout 1:1 to about 20:1, preferably in a ratio of about 1:1 to about12:1, and more preferably in a ratio of about 2:1 to about 6:1.Additionally, the overall concentration of siNA in solution willtypically be from about 25 μg/mL to about 1 mg/mL, preferably from about25 μg/mL to about 500 μg/mL, and more preferably from about 100 μg/mL toabout 250 μg/mL. The combination of the molecules of interest andcationic lipids in detergent solution is kept, typically at roomtemperature, for a period of time which is sufficient for the coatedcomplexes to form. Alternatively, the molecules of interest and cationiclipids can be combined in the detergent solution and warmed totemperatures of up to about 37° C. For molecules (e.g., certainpolynucleotides herein) which are particularly sensitive to temperature,the coated complexes can be formed at lower temperatures, typically downto about 4° C.

In one embodiment, the biologically active molecule to lipid ratios(mass/mass ratios) in a formed formulation or composition range fromabout 0.01 to about 0.08. The ratio of the starting materials also fallswithin this range because the purification step typically removes theunencapsulated biologically active molecule as well as the emptyliposomes. In another embodiment, the formulated biologically activemolecule composition preparation uses about 400 μg siNA per 10 mg totallipid or a biologically active molecule to lipid ratio of about 0.01 toabout 0.08 and, more preferably, about 0.04, which corresponds to 1.25mg of total lipid per 50 μg of biologically active molecule. Aformulation or composition of the invention is developed to targetspecific organs, tissues, or cell types. In one embodiment, aformulation or composition of the invention is developed to target theliver or hepatocytes. Ratios of the various components of theformulation or composition are adjusted to target specific organs,tissues, or cell types.

In one embodiment, the invention features a method for delivering oradministering a biologically active molecule to a cell or cells in asubject or organism, comprising administering a formulation orcomposition of the invention under conditions suitable for delivery ofthe biologically active molecule component of the formulation orcomposition to the cell or cells of the subject or organism. In oneembodiment, the formulation or composition is contacted with the cell orcells of the subject or organism as is generally known in the art, suchas via parental administration (e.g., intravenous, intramuscular,subcutaneous administration) or pulmonary administration of theformulation or composition with or without excipients to facilitate theadministration.

In one embodiment, the invention features a method for delivering oradministering a biologically active molecule to liver or liver cells(e.g., hepatocytes) in a subject or organism, comprising administering aformulation or composition of the invention under conditions suitablefor delivery of the biologically active molecule component of theformulation or composition to the liver or liver cells (e.g.,hepatocytes) of the subject or organism. In one embodiment, theformulation or composition is contacted with the liver or liver cells ofthe subject or organism as is generally known in the art, such as viaparental administration (e.g., intravenous, intramuscular, subcutaneousadministration) or local administration (e.g., direct injection, portalvein injection, catheterization, stenting etc.) of the formulation orcomposition with or without excipients to facilitate the administration.

In one embodiment, the invention features a method for delivering oradministering a biologically active molecule to kidney or kidney cellsin a subject or organism, comprising administering a formulation orcomposition of the invention under conditions suitable for delivery ofthe biologically active molecule component of the formulation orcomposition to the kidney or kidney cells of the subject or organism. Inone embodiment, the formulation or composition is contacted with thekidney or kidney cells of the subject or organism as is generally knownin the art, such as via parental administration (e.g., intravenous,intramuscular, subcutaneous administration) or local administration(e.g., direct injection, catheterization, stenting etc.) of theformulation or composition with or without excipients to facilitate theadministration.

In one embodiment, the invention features a method for delivering oradministering a biologically active molecule to tumor or tumor cells ina subject or organism, comprising administering a formulation orcomposition of the invention under conditions suitable for delivery ofthe biologically active molecule component of the formulation orcomposition to the tumor or tumor cells of the subject or organism. Inone embodiment, the formulation or composition is contacted with thetumor or tumor cells of the subject or organism as is generally known inthe art, such as via parental administration (e.g., intravenous,intramuscular, subcutaneous administration) or local administration(e.g., direct injection, catheterization, stenting etc.) of theformulation or composition with or without excipients to facilitate theadministration.

In one embodiment, the invention features a method for delivering oradministering a biologically active molecule to CNS or CNS cells (e.g.,brain, spinal cord) in a subject or organism, comprising administering aformulation or composition of the invention under conditions suitablefor delivery of the biologically active molecule component of theformulation or composition to the CNS or CNS cells of the subject ororganism. In one embodiment, the formulation or composition is contactedwith the CNS or CNS cells of the subject or organism as is generallyknown in the art, such as via parental administration (e.g.,intravenous, intramuscular, subcutaneous administration) or localadministration (e.g., direct injection, catheterization, stenting etc.)of the formulation or composition with or without excipients tofacilitate the administration.

In one embodiment, the invention features a method for delivering oradministering a biologically active molecule to lung or lung cells in asubject or organism, comprising administering a formulation orcomposition of the invention under conditions suitable for delivery ofthe biologically active molecule component of the formulation orcomposition to the lung or lung cells of the subject or organism. In oneembodiment, the formulation or composition is contacted with the lung orlung cells of the subject or organism as is generally known in the art,such as via parental administration (e.g., intravenous, intramuscular,subcutaneous administration) or local administration (e.g., pulmonaryadministration directly to lung tissues and cells) of the formulation orcomposition with or without excipients to facilitate the administration.

In one embodiment, the invention features a method for delivering oradministering a biologically active molecule to vascular or vascularcells in a subject or organism, comprising administering a formulationor composition of the invention under conditions suitable for deliveryof the biologically active molecule component of the formulation orcomposition to the vascular or vascular cells of the subject ororganism. In one embodiment, the formulation or composition is contactedwith the vascular or vascular cells of the subject or organism as isgenerally known in the art, such as via parental administration (e.g.,intravenous, intramuscular, subcutaneous administration) or localadministration (e.g., clamping, catheterization, stenting etc.) of theformulation or composition with or without excipients to facilitate theadministration.

In one embodiment, the invention features a method for delivering oradministering a biologically active molecule to skin or skin cells(e.g., dermis or dermis cells, follicle or follicular cells) in asubject or organism, comprising administering a formulation orcomposition of the invention under conditions suitable for delivery ofthe biologically active molecule component of the formulation orcomposition to the skin or skin cells of the subject or organism. In oneembodiment, the formulation or composition is contacted with the skin orskin cells of the subject or organism as is generally known in the art,such as via parental administration (e.g., intravenous, intramuscular,subcutaneous administration) or local administration (e.g., directdermal application, iontophoresis etc.) of the formulation orcomposition with or without excipients to facilitate the administration.

In one embodiment, the invention features a method for delivering oradministering a biologically active molecule to the eye or ocular cells(e.g., macula, fovea, cornea, retina etc.) in a subject or organism,comprising administering a formulation or composition of the inventionunder conditions suitable for delivery of the biologically activemolecule component of the formulation or composition to the eye orocular cells of the subject or organism. In one embodiment, theformulation or composition is contacted with the eye or ocular cells ofthe subject or organism as is generally known in the art, such as viaparental administration (e.g., intravenous, intramuscular, subcutaneousadministration) or local administration (e.g., direct injection,intraocular injection, periocular injection, iontophoresis, use ofeyedrops, inplants etc.) of the formulation or composition with orwithout excipients to facilitate the administration.

In one embodiment, the invention features a method for delivering oradministering a biologically active molecule to the ear or cells of theear (e.g., inner ear, middle ear, outer ear) in a subject or organism,comprising administering a formulation or composition of the inventionunder conditions suitable for delivery of the biologically activemolecule component of the formulation or composition to the ear or earcells of the subject or organism. In one embodiment, the administrationcomprises methods and devices as described in U.S. Pat. Nos. 5,421,818,5,476,446, 5,474,529, 6,045,528, 6,440,102, 6,685,697, 6,120,484; and5,572,594; all incorporated by reference in their entireties herein andthe teachings of Silverstein, 1999, Ear Nose Throat J., 78, 595-8, 600;and Jackson and Silverstein, 2002, Otolaryngol Clin North Am., 35,639-53, and adapted for use the compositions of the invention.

In one embodiment, the invention features a formulated siNA compositioncomprising a short interfering nucleic acid (siNA) molecule thatdown-regulates expression of a target gene, wherein said siNA moleculecomprises about 15 to about 28 base pairs.

In one embodiment, the invention features a formulated siNA compositioncomprising a double stranded short interfering nucleic acid (siNA)molecule that directs cleavage of a target RNA via RNA interference(RNAi), wherein the double stranded siNA molecule comprises a first anda second strand, each strand of the siNA molecule is about 18 to about28 nucleotides in length, the first strand of the siNA comprisesnucleotide sequence having sufficient complementarity to the target RNAfor the siNA molecule to direct cleavage of the target RNA via RNAinterference, and the second strand of said siNA molecule comprisesnucleotide sequence that is complementary to the first strand.

In one embodiment, the invention features a formulated siNA compositioncomprising a double stranded short interfering nucleic acid (siNA)molecule that directs cleavage of a target RNA via RNA interference(RNAi), wherein the double stranded siNA molecule comprises a first anda second strand, each strand of the siNA molecule is about 18 to about23 nucleotides in length, the first strand of the siNA moleculecomprises nucleotide sequence having sufficient complementarity to thetarget RNA for the siNA molecule to direct cleavage of the target RNAvia RNA interference, and the second strand of said siNA moleculecomprises nucleotide sequence that is complementary to the first strand.

In one embodiment, the invention features a formulated siNA compositioncomprising a chemically synthesized double stranded short interferingnucleic acid (siNA) molecule that directs cleavage of a target RNA viaRNA interference (RNAi), wherein each strand of the siNA molecule isabout 18 to about 28 nucleotides in length; and one strand of the siNAmolecule comprises nucleotide sequence having sufficient complementarityto the target RNA for the siNA molecule to direct cleavage of the targetRNA via RNA interference.

In one embodiment, the invention features a formulated siNA compositioncomprising a chemically synthesized double stranded short interferingnucleic acid (siNA) molecule that directs cleavage of a target RNA viaRNA interference (RNAi), wherein each strand of the siNA molecule isabout 18 to about 23 nucleotides in length; and one strand of the siNAmolecule comprises nucleotide sequence having sufficient complementarityto the target RNA for the siNA molecule to direct cleavage of the targetRNA via RNA interference.

In one embodiment, the invention features a formulated siNA compositioncomprising a siNA molecule that down-regulates expression of a targetgene, for example, wherein the target gene comprises target encodingsequence. In one embodiment, the invention features a siNA molecule thatdown-regulates expression of a target gene, for example, wherein thetarget gene comprises target non-coding sequence or regulatory elementsinvolved in target gene expression.

In one embodiment, a siNA of the invention is used to inhibit theexpression of target genes or a target gene family, wherein the genes orgene family sequences share sequence homology. Such homologous sequencescan be identified as is known in the art, for example using sequencealignments. siNA molecules can be designed to target such homologoussequences, for example using perfectly complementary sequences or byincorporating non-canonical base pairs, for example mismatches and/orwobble base pairs that can provide additional target sequences. Ininstances where mismatches are identified, non-canonical base pairs (forexample, mismatches and/or wobble bases) can be used to generate siNAmolecules that target more than one gene sequence. In a non-limitingexample, non-canonical base pairs such as UU and CC base pairs are usedto generate siNA molecules that are capable of targeting sequences fordiffering targets that share sequence homology. As such, one advantageof using siNAs of the invention is that a single siNA can be designed toinclude nucleic acid sequence that is complementary to the nucleotidesequence that is conserved between the homologous genes. In thisapproach, a single siNA can be used to inhibit expression of more thanone gene instead of using more than one siNA molecule to target thedifferent genes.

In one embodiment, the invention features a formulated siNA compositioncomprising a siNA molecule having RNAi activity against a target RNA,wherein the siNA molecule comprises a sequence complementary to any RNAhaving target encoding sequence. Examples of siNA molecules suitable forthe formulations described herein are provided in InternationalApplication Serial Number US04/106390 (WO 05/19453), which is herebyincorporated by reference in its entirety. Chemical modifications asdescribed in PCT/US 2004/106390 (WO 05/19453), U.S. Ser. No. 10/444,853,filed May 23, 2003 U.S. Ser. No. 10/923,536 filed Aug. 20, 2004, U.S.Ser. No. 11/234,730, filed Sep. 23, 2005 or U.S. Ser. No. 11/299,254,filed Dec. 8, 2005, all incorporated by reference in their entiretiesherein, or otherwise described herein can be applied to any siNAconstruct of the invention. In another embodiment, a siNA molecule ofthe invention includes a nucleotide sequence that can interact withnucleotide sequence of a target gene and thereby mediate silencing oftarget gene expression, for example, wherein the siNA mediatesregulation of target gene expression by cellular processes that modulatethe chromatin structure or methylation patterns of the target gene andprevent transcription of the target gene.

In one embodiment, siNA molecules of the invention are used to downregulate or inhibit the expression of target proteins arising fromtarget haplotype polymorphisms that are associated with a disease orcondition (e.g. alopecia, hair loss, and/or atrichia). Analysis oftarget genes, or target protein or RNA levels can be used to identifysubjects with such polymorphisms or those subjects who are at risk ofdeveloping traits, conditions, or diseases described herein. Thesesubjects are amenable to treatment, for example, treatment with siNAmolecules of the invention and any other composition useful in treatingdiseases related to target gene expression. As such, analysis of targetprotein or RNA levels can be used to determine treatment type and thecourse of therapy in treating a subject. Monitoring of target protein orRNA levels can be used to predict treatment outcome and to determine theefficacy of compounds and compositions that modulate the level and/oractivity of certain target proteins associated with a trait, condition,or disease.

In one embodiment, a siNA molecule of the invention comprises anantisense strand comprising a nucleotide sequence that is complementaryto a nucleotide sequence or a portion thereof encoding a target protein.The siNA further comprises a sense strand, wherein said sense strandcomprises a nucleotide sequence of a target gene or a portion thereof.

In another embodiment, a siNA of the invention comprises an antisenseregion comprising a nucleotide sequence that is complementary to anucleotide sequence encoding a target protein or a portion thereof. ThesiNA molecule further comprises a sense region, wherein said senseregion comprises a nucleotide sequence of a target gene or a portionthereof.

In another embodiment, a siNA of the invention comprises a nucleotidesequence in the antisense region of the siNA molecule that iscomplementary to a nucleotide sequence or portion of sequence of atarget gene. In another embodiment, a siNA of the invention comprises aregion, for example, the antisense region of the siNA construct that iscomplementary to a sequence comprising a target gene sequence or aportion thereof.

In one embodiment, a siNA molecule of the invention comprises anantisense strand having about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides,wherein the antisense strand is complementary to a RNA sequence or aportion thereof encoding a target protein, and wherein said siNA furthercomprises a sense strand having about 15 to about 30 (e.g., about 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, and wherein said sense strand and said antisense strand aredistinct nucleotide sequences where at least about 15 nucleotides ineach strand are complementary to the other strand.

In another embodiment, a siNA molecule of the invention comprises anantisense region having about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides,wherein the antisense region is complementary to a RNA sequence encodinga target protein, and wherein said siNA further comprises a sense regionhaving about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein said senseregion and said antisense region are comprised in a linear moleculewhere the sense region comprises at least about 15 nucleotides that arecomplementary to the antisense region.

In one embodiment, a siNA molecule of the invention has RNAi activitythat modulates expression of RNA encoded by a target gene. Becausetarget genes can share some degree of sequence homology with each other,siNA molecules can be designed to target a class of target genes oralternately specific target genes (e.g., polymorphic variants) byselecting sequences that are either shared amongst different targets oralternatively that are unique for a specific target. Therefore, in oneembodiment, the siNA molecule can be designed to target conservedregions of target RNA sequences having homology among several targetgene variants so as to target a class of target genes with one siNAmolecule. Accordingly, in one embodiment, the siNA molecule of theinvention modulates the expression of one or both target alleles in asubject. In another embodiment, the siNA molecule can be designed totarget a sequence that is unique to a specific target RNA sequence(e.g., a single target allele or target single nucleotide polymorphism(SNP)) due to the high degree of specificity that the siNA moleculerequires to mediate RNAi activity.

In one embodiment, a siNA molecule of the invention is double-stranded.In another embodiment, the siNA molecules of the invention consist ofduplex nucleic acid molecules containing about 15 to about 30 base pairsbetween oligonucleotides comprising about 15 to about 30 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides. In yet another embodiment, siNA molecules of the inventioncomprise duplex nucleic acid molecules with overhanging ends of about 1to about 3 (e.g., about 1, 2, or 3) nucleotides, for example, about21-nucleotide duplexes with about 19 base pairs and 3′-terminalmononucleotide, dinucleotide, or trinucleotide overhangs. In yet anotherembodiment, siNA molecules of the invention comprise duplex nucleic acidmolecules with blunt ends, where both ends are blunt, or alternatively,where one of the ends is blunt.

In one embodiment, siNA molecules of the invention have specificity fornucleic acid molecules expressing target proteins, such as RNA encodinga target protein. In one embodiment, a siNA molecule of the invention isRNA based (e.g., a siNA comprising 2′-OH nucleotides) and includes oneor more chemical modifications, such as those described herein.Non-limiting examples of such chemical modifications include withoutlimitation phosphorothioate internucleotide linkages,2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluororibonucleotides, “universal base” nucleotides, “acyclic” nucleotides,5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxyabasic residue incorporation. These chemical modifications, when used invarious siNA constructs, (e.g., RNA based siNA constructs), are shown topreserve RNAi activity in cells while at the same time, dramaticallyincreasing the serum stability of these compounds. Furthermore, contraryto the data published by Parrish et al., supra, applicant demonstratesthat multiple (greater than one) phosphorothioate substitutions arewell-tolerated and confer substantial increases in serum stability formodified siNA constructs.

In one embodiment, a siNA molecule of the invention comprises modifiednucleotides while maintaining the ability to mediate RNAi. The modifiednucleotides can be used to improve in vitro or in vivo characteristicssuch as stability, activity, and/or bioavailability. For example, a siNAmolecule of the invention can comprise modified nucleotides as apercentage of the total number of nucleotides present in the siNAmolecule. As such, a siNA molecule of the invention can generallycomprise about 5% to about 100% modified nucleotides (e.g., about 5%,10%, 15%,20%,25%,30%,35%,40%,45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%,90%, 95% or 100% modified nucleotides). The actual percentage ofmodified nucleotides present in a given siNA molecule will depend on thetotal number of nucleotides present in the siNA. If the siNA molecule issingle stranded, the percent modification can be based upon the totalnumber of nucleotides present in the single stranded siNA molecules.Likewise, if the siNA molecule is double stranded, the percentmodification can be based upon the total number of nucleotides presentin the sense strand, antisense strand, or both the sense and antisensestrands.

One aspect of the invention features a formulated siNA compositioncomprising a double-stranded short interfering nucleic acid (siNA)molecule that down-regulates expression of a target gene. In oneembodiment, the double stranded siNA molecule comprises one or morechemical modifications and each strand of the double-stranded siNA isabout 21 nucleotides long. In one embodiment, the double-stranded siNAmolecule does not contain any ribonucleotides. In another embodiment,the double-stranded siNA molecule comprises one or more ribonucleotides.In one embodiment, each strand of the double-stranded siNA moleculeindependently comprises about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides,wherein each strand comprises about 15 to about 30 (e.g., about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotidesthat are complementary to the nucleotides of the other strand. In oneembodiment, one of the strands of the double-stranded siNA moleculecomprises a nucleotide sequence that is complementary to a nucleotidesequence or a portion thereof of the target gene, and the second strandof the double-stranded siNA molecule comprises a nucleotide sequencesubstantially similar to the nucleotide sequence of the target gene or aportion thereof.

In another embodiment, the invention features a formulated siNAcomposition comprising a double-stranded short interfering nucleic acid(siNA) molecule that down-regulates expression of a target genecomprising an antisense region, wherein the antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence ofthe target gene or a portion thereof, and a sense region, wherein thesense region comprises a nucleotide sequence substantially similar tothe nucleotide sequence of the target gene or a portion thereof. In oneembodiment, the antisense region and the sense region independentlycomprise about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein theantisense region comprises about 15 to about 30 (e.g. about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides thatare complementary to nucleotides of the sense region.

In another embodiment, the invention features a formulated siNAcomposition comprising a double-stranded short interfering nucleic acid(siNA) molecule that down-regulates expression of a target genecomprising a sense region and an antisense region, wherein the antisenseregion comprises a nucleotide sequence that is complementary to anucleotide sequence of RNA encoded by the target gene or a portionthereof and the sense region comprises a nucleotide sequence that iscomplementary to the antisense region.

In one embodiment, a siNA molecule of the invention comprises bluntends, i.e., ends that do not include any overhanging nucleotides. Forexample, a siNA molecule-comprising modifications described in U.S. Ser.No. 10/444,853, filed May 23, 2003, U.S. Ser. No. 10/923,536 filed Aug.20, 2004, or U.S. Ser. No. 11/234,730, filed Sep. 23, 2005, allincorporated by reference in their entireties herein, or any combinationthereof and/or any length described herein can comprise blunt ends orends with no overhanging nucleotides.

In one embodiment, any siNA molecule of the invention can comprise oneor more blunt ends, i.e. where a blunt end does not have any overhangingnucleotides. In one embodiment, the blunt ended siNA molecule has anumber of base pairs equal to the number of nucleotides present in eachstrand of the siNA molecule. In another embodiment, the siNA moleculecomprises one blunt end, for example wherein the 5′-end of the antisensestrand and the 3′-end of the sense strand do not have any overhangingnucleotides. In another example, the siNA molecule comprises one bluntend, for example wherein the 3′-end of the antisense strand and the5′-end of the sense strand do not have any overhanging nucleotides. Inanother example, a siNA molecule comprises two blunt ends, for examplewherein the 3′-end of the antisense strand and the 5′-end of the sensestrand as well as the 5′-end of the antisense strand and 3′-end of thesense strand do not have any overhanging nucleotides. A blunt ended siNAmolecule can comprise, for example, from about 15 to about 30nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 nucleotides). Other nucleotides present in a bluntended siNA molecule can comprise, for example, mismatches, bulges,loops, or wobble base pairs to modulate the activity of the siNAmolecule to mediate RNA interference.

By “blunt ends” is meant symmetric termini, or termini of a doublestranded siNA molecule having no overhanging nucleotides. The twostrands of a double stranded siNA molecule align with each other withoutover-hanging nucleotides at the termini. For example, a blunt ended siNAconstruct comprises terminal nucleotides that are complementary betweenthe sense and antisense regions of the siNA molecule.

In one embodiment, the invention features a formulated siNA compositioncomprising a double-stranded short interfering nucleic acid (siNA)molecule that down-regulates expression of a target gene, wherein thesiNA molecule is assembled from two separate oligonucleotide fragmentswherein one fragment comprises the sense region and the second fragmentcomprises the antisense region of the siNA molecule. The sense regioncan be connected to the antisense region via a linker molecule, such asa polynucleotide linker or a non-nucleotide linker.

In one embodiment, the invention features a formulated siNA compositioncomprising a double-stranded short interfering nucleic acid (siNA)molecule that down-regulates expression of a target gene, wherein thesiNA molecule comprises about 15 to about 30 (e.g. about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, andwherein each strand of the siNA molecule comprises one or more chemicalmodifications. In another embodiment, one of the strands of thedouble-stranded siNA molecule comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of a target gene or a portionthereof, and the second strand of the double-stranded siNA moleculecomprises a nucleotide sequence substantially similar to the nucleotidesequence or a portion thereof of the target gene. In another embodiment,one of the strands of the double-stranded siNA molecule comprises anucleotide sequence that is complementary to a nucleotide sequence of atarget gene or portion thereof, and the second strand of thedouble-stranded siNA molecule comprises a nucleotide sequencesubstantially similar to the nucleotide sequence or portion thereof ofthe target gene. In another embodiment, each strand of the siNA moleculecomprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, and each strandcomprises at least about 15 to about 30 (e.g. about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that arecomplementary to the nucleotides of the other strand.

In any of the embodiments described herein, a siNA molecule of theinvention can comprise no ribonucleotides. Alternatively, a siNAmolecule of the invention can comprise one or more ribonucleotides.

In one embodiment, a siNA molecule of the invention comprises anantisense region comprising a nucleotide sequence that is complementaryto a nucleotide sequence of a target gene or a portion thereof, and thesiNA further comprises a sense region comprising a nucleotide sequencesubstantially similar to the nucleotide sequence of the target gene or aportion thereof. In another embodiment, the antisense region and thesense region each comprise about 15 to about 30 (e.g. about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides andthe antisense region comprises at least about 15 to about 30 (e.g. about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides that are complementary to nucleotides of the sense region.The target gene can comprise, for example, sequences referred to byGenbank Accession Nos. in PCT Publication No. WO 03/74654, serial No.PCT/US03/05028 or U.S. Ser. No. 10/923,536. In another embodiment, thesiNA is a double stranded nucleic acid molecule, where each of the twostrands of the siNA molecule independently comprise about 15 to about 40(e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides, and whereone of the strands of the siNA molecule comprises at least about 15(e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 or more)nucleotides that are complementary to the nucleic acid sequence of thetarget gene or a portion thereof.

In one embodiment, a siNA molecule of the invention comprises a senseregion and an antisense region, wherein the antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence ofRNA encoded by a target gene, or a portion thereof, and the sense regioncomprises a nucleotide sequence that is complementary to the antisenseregion. In one embodiment, the siNA molecule is assembled from twoseparate oligonucleotide fragments, wherein one fragment comprises thesense region and the second fragment comprises the antisense region ofthe siNA molecule. In another embodiment, the sense region is connectedto the antisense region via a linker molecule. In another embodiment,the sense region is connected to the antisense region via a linkermolecule, such as a nucleotide or non-nucleotide linker. The target genecan comprise, for example, sequences referred to in PCT Publication No.WO 03/74654, serial No. PCT/US03/05028 or U.S. Ser. No. 10/923,536 orotherwise known in the art.

In one embodiment, the invention features a formulated siNA compositioncomprising a double-stranded short interfering nucleic acid (siNA)molecule that down-regulates expression of a target gene comprising asense region and an antisense region, wherein the antisense regioncomprises a nucleotide sequence that is complementary to a nucleotidesequence of RNA encoded by the target gene or a portion thereof and thesense region comprises a nucleotide sequence that is complementary tothe antisense region, and wherein the siNA molecule has one or moremodified pyrimidine and/or purine nucleotides. In one embodiment, thepyrimidine nucleotides in the sense region are 2′-O-methylpyrimidinenucleotides or 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purinenucleotides present in the sense region are 2′-deoxy purine nucleotides.In another embodiment, the pyrimidine nucleotides in the sense regionare 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotidespresent in the sense region are 2′-O-methyl purine nucleotides. Inanother embodiment, the pyrimidine nucleotides in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotidespresent in the sense region are 2′-deoxy purine nucleotides. In oneembodiment, the pyrimidine nucleotides in the antisense region are2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotidespresent in the antisense region are 2′-O-methyl or 2′-deoxy purinenucleotides. In another embodiment of any of the above-described siNAmolecules, any nucleotides present in a non-complementary region of thesense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the invention features a formulated siNA compositioncomprising a double-stranded short interfering nucleic acid (siNA)molecule that down-regulates expression of a target gene, wherein thesiNA molecule is assembled from two separate oligonucleotide fragmentswherein one fragment comprises the sense region and the second fragmentcomprises the antisense region of the siNA molecule, and wherein thefragment comprising the sense region includes a terminal cap moiety atthe 5′-end, the 3′-end, or both of the 5′ and 3′ ends of the fragment.In one embodiment, the terminal cap moiety is an inverted deoxy abasicmoiety or glyceryl moiety. In one embodiment, each of the two fragmentsof the siNA molecule independently comprise about 15 to about 30 (e.g.about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides. In another embodiment, each of the two fragments of thesiNA molecule independently comprise about 15 to about 40 (e.g. about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23,33, 34, 35, 36, 37, 38, 39, or 40) nucleotides. In a non-limitingexample, each of the two fragments of the siNA molecule comprises about21 nucleotides.

In one embodiment, the invention features a formulated siNA compositioncomprising a siNA molecule comprising at least one modified nucleotide,wherein the modified nucleotide is a 2′-deoxy-2′-fluoro nucleotide. ThesiNA can be, for example, about 15 to about 40 nucleotides in length. Inone embodiment, all pyrimidine nucleotides present in the siNA are2′-deoxy-2′-fluoro pyrimidine nucleotides. In one embodiment, themodified nucleotides in the siNA include at least one 2′-deoxy-2′-fluorocytidine or 2′-deoxy-2′-fluoro uridine nucleotide. In anotherembodiment, the modified nucleotides in the siNA include at least one2′-fluoro cytidine and at least one 2′-deoxy-2′-fluoro uridinenucleotides. In one embodiment, all uridine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, allcytidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidinenucleotides. In one embodiment, all adenosine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In one embodiment,all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroguanosine nucleotides. The siNA can further comprise at least onemodified internucleotidic linkage, such as phosphorothioate linkage. Inone embodiment, the 2′-deoxy-2′-fluoronucleotides are present atspecifically selected locations in the siNA that are sensitive tocleavage by ribonucleases, such as locations having pyrimidinenucleotides.

In one embodiment, the invention features a method of increasing thestability of a siNA molecule of the invention against cleavage byribonucleases comprising introducing at least one modified nucleotideinto the siNA molecule, wherein the modified nucleotide is a2′-deoxy-2′-fluoro nucleotide. In one embodiment, all pyrimidinenucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidinenucleotides. In one embodiment, the modified nucleotides in the siNAinclude at least one 2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluorouridine nucleotide. In another embodiment, the modified nucleotides inthe siNA include at least one 2′-fluoro cytidine and at least one2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all uridinenucleotides present in the siNA are 2′-deoxy-2′-fluoro uridinenucleotides. In one embodiment, all cytidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In one embodiment, alladenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroadenosine nucleotides. In one embodiment, all guanosine nucleotidespresent in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. ThesiNA can further comprise at least one modified internucleotidiclinkage, such as phosphorothioate linkage. In one embodiment, the2′-deoxy-2′-fluoronucleotides are present at specifically selectedlocations in the siNA that are sensitive to cleavage by ribonucleases,such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a formulated siNA compositioncomprising a double-stranded short interfering nucleic acid (siNA)molecule that down-regulates expression of a target gene comprising asense region and an antisense region, wherein the antisense regioncomprises a nucleotide sequence that is complementary to a nucleotidesequence of RNA encoded by the target gene or a portion thereof and thesense region comprises a nucleotide sequence that is complementary tothe antisense region, and wherein the purine nucleotides present in theantisense region comprise 2′-deoxy-purine nucleotides. In an alternativeembodiment, the purine nucleotides present in the antisense regioncomprise 2′-O-methyl purine nucleotides. In either of the aboveembodiments, the antisense region can comprise a phosphorothioateinternucleotide linkage at the 3′ end of the antisense region.Alternatively, in either of the above embodiments, the antisense regioncan comprise a glyceryl modification at the 3′ end of the antisenseregion. In another embodiment of any of the above-described siNAmolecules, any nucleotides present in a non-complementary region of theantisense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the antisense region of a siNA molecule of theinvention comprises sequence complementary to a portion of a targettranscript having sequence unique to a particular target disease relatedallele, such as sequence comprising a single nucleotide polymorphism(SNP) associated with the disease specific allele. As such, theantisense region of a siNA molecule of the invention can comprisesequence complementary to sequences that are unique to a particularallele to provide specificity in mediating selective RNAi against thedisease, condition, or trait related allele.

In one embodiment, the invention features a formulated siNA compositioncomprising a double-stranded short interfering nucleic acid (siNA)molecule that down-regulates expression of a target gene, wherein thesiNA molecule is assembled from two separate oligonucleotide fragmentswherein one fragment comprises the sense region and the second fragmentcomprises the antisense region of the siNA molecule. In anotherembodiment, the siNA molecule is a double stranded nucleic acidmolecule, where each strand is about 21 nucleotides long and where about19 nucleotides of each fragment of the siNA molecule are base-paired tothe complementary nucleotides of the other fragment of the siNAmolecule, wherein at least two 3′ terminal nucleotides of each fragmentof the siNA molecule are not base-paired to the nucleotides of the otherfragment of the siNA molecule. In another embodiment, the siNA moleculeis a double stranded nucleic acid molecule, where each strand is about19 nucleotide long and where the nucleotides of each fragment of thesiNA molecule are base-paired to the complementary nucleotides of theother fragment of the siNA molecule to form at least about 15 (e.g., 15,16, 17, 18, or 19) base pairs, wherein one or both ends of the siNAmolecule are blunt ends. In one embodiment, each of the two 3′ terminalnucleotides of each fragment of the siNA molecule is a2′-deoxy-pyrimidine nucleotide, such as a 2′-deoxy-thymidine. In anotherembodiment, all nucleotides of each fragment of the siNA molecule arebase-paired to the complementary nucleotides of the other fragment ofthe siNA molecule. In another embodiment, the siNA molecule is a doublestranded nucleic acid molecule of about 19 to about 25 base pairs havinga sense region and an antisense region, where about 19 nucleotides ofthe antisense region are base-paired to the nucleotide sequence or aportion thereof of the RNA encoded by the target gene. In anotherembodiment, about 21 nucleotides of the antisense region are base-pairedto the nucleotide sequence or a portion thereof of the RNA encoded bythe target gene. In any of the above embodiments, the 5′-end of thefragment comprising said antisense region can optionally include aphosphate group.

In any of the embodiments described herein, a siNA molecule of theinvention can comprise one or more of the stabilization chemistriesshown in Table I or described in PCT/US 2004/106390 (WO 05/19453), U.S.Ser. No. 10/444,853, filed May 23, 2003 U.S. Ser. No. 10/923,536 filedAug. 20, 2004, U.S. Ser. No. 11/234,730, filed Sep. 23, 2005 or U.S.Ser. No. 11/299,254, filed Dec. 8, 2005, all incorporated by referencein their entireties herein.

In one embodiment, the invention features a formulated siNA compositioncomprising a double-stranded short interfering nucleic acid (siNA)molecule that inhibits the expression of a target RNA sequence (e.g.,wherein said target RNA sequence is encoded by a target gene involved inthe target pathway), wherein the siNA molecule does not contain anyribonucleotides and wherein each strand of the double-stranded siNAmolecule is about 15 to about 30 nucleotides. In one embodiment, thesiNA molecule is 21 nucleotides in length. Examples ofnon-ribonucleotide containing siNA constructs are combinations ofstabilization chemistries described in PCT/US 2004/106390 (WO 05/19453),U.S. Ser. No. 10/444,853, filed May 23, 2003 U.S. Ser. No. 10/923,536filed Aug. 20, 2004, U.S. Ser. No. 11/234,730, filed Sep. 23, 2005 orU.S. Ser. No. 11/299,254, filed Dec. 8, 2005, all incorporated byreference in their entireties herein.

In one embodiment, the invention features a formulated siNA compositioncomprising a chemically synthesized double stranded RNA molecule thatdirects cleavage of a target RNA via RNA interference, wherein eachstrand of said RNA molecule is about 15 to about 30 nucleotides inlength; one strand of the RNA molecule comprises nucleotide sequencehaving sufficient complementarity to the target RNA for the RNA moleculeto direct cleavage of the target RNA via RNA interference; and whereinat least one strand of the RNA molecule optionally comprises one or morechemically modified nucleotides described herein, such as withoutlimitation deoxynucleotides, 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoronucleotides, 2′-O-methoxyethyl nucleotides etc.

In one embodiment, the invention features a composition comprising aformulated siNA composition of the invention in a pharmaceuticallyacceptable carrier or diluent.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits the expression ofa target RNA sequence, wherein the siNA molecule does not contain anyribonucleotides and wherein each strand of the double-stranded siNAmolecule is about 15 to about 30 nucleotides. In one embodiment, thesiNA molecule is 21 nucleotides in length. Examples ofnon-ribonucleotide containing siNA constructs are combinations ofstabilization chemistries shown in Table I in any combination ofSense/Antisense chemistries, such as Stab 7/8, Stab 7/11, Stab 8/8, Stab18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab 18/13, Stab 7/19, Stab8/19, Stab 18/19, Stab 7/20, Stab 8/20, Stab 18/20, Stab 7/32, Stab8/32, or Stab 18/32 (e.g., any siNA having Stab 7, 8, 11, 12, 13, 14,15, 17, 18, 19, 20, or 32 sense or antisense strands or any combinationthereof). Herein, numeric Stab chemistries can include both 2′-fluoroand 2′-OCF3 versions of the chemistries shown in Table I. For example,“Stab 7/8” refers to both Stab 7/8 and Stab 7F/8F etc. In oneembodiment, the invention features a chemically synthesized doublestranded RNA molecule that directs cleavage of a target RNA via RNAinterference, wherein each strand of said RNA molecule is about 15 toabout 30 nucleotides in length; one strand of the RNA molecule comprisesnucleotide sequence having sufficient complementarity to the target RNAfor the RNA molecule to direct cleavage of the target RNA via RNAinterference; and wherein at least one strand of the RNA moleculeoptionally comprises one or more chemically modified nucleotidesdescribed herein, such as without limitation deoxynucleotides,2′-O-methyl nucleotides, 2′-deoxy-2′-fluoro nucleotides,2′-O-methoxyethyl nucleotides, 4′-thio nucleotides, 2′-O-trifluoromethylnucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides,2′-O-difluoromethoxy-ethoxy nucleotides, etc.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises one or more (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides comprising a backbonemodified internucleotide linkage having Formula I:

wherein each R1 and R2 is independently any nucleotide, non-nucleotide,or polynucleotide which can be naturally-occurring orchemically-modified, each X and Y is independently O, S, N, alkyl, orsubstituted alkyl, each Z and W is independently O, S, N, alkyl,substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or acetyl andwherein W, X, Y, and Z are optionally not all O. In another embodiment,a backbone modification of the invention comprises a phosphonoacetateand/or thiophosphonoacetate internucleotide linkage (see for exampleSheehan et al., 2003, Nucleic Acids Research, 31, 4109-4118).

The chemically-modified internucleotide linkages having Formula I, forexample, wherein any Z, W, X, and/or Y independently comprises a sulphuratom, can be present in one or both oligonucleotide strands of the siNAduplex, for example, in the sense strand, the antisense strand, or bothstrands. The siNA molecules of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically-modifiedinternucleotide linkages having Formula I at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the sense strand, the antisense strand, orboth strands. For example, an exemplary siNA molecule of the inventioncan comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, ormore) chemically-modified internucleotide linkages having Formula I atthe 5′-end of the sense strand, the antisense strand, or both strands.In another non-limiting example, an exemplary siNA molecule of theinvention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) pyrimidine nucleotides with chemically-modifiedinternucleotide linkages having Formula I in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotideswith chemically-modified internucleotide linkages having Formula I inthe sense strand, the antisense strand, or both strands. In anotherembodiment, a siNA molecule of the invention having internucleotidelinkage(s) of Formula I also comprises a chemically-modified nucleotideor non-nucleotide having any of Formulae I-VII.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises one or more (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotideshaving Formula II:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalklylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be complementary or non-complementary to targetRNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole,5-nitroindole, nebularine, pyridone, pyridinone, or any othernon-naturally occurring universal base that can be complementary ornon-complementary to target RNA. In one embodiment, R3 and/or R7comprises a conjugate moiety and a linker (e.g., a nucleotide ornon-nucleotide linker as described herein or otherwise known in theart). Non-limiting examples of conjugate moieties include ligands forcellular receptors, such as peptides derived from naturally occurringprotein ligands; protein localization sequences, including cellular ZIPcode sequences; antibodies; nucleic acid aptamers; vitamins and otherco-factors, such as folate and N-acetylgalactosamine; polymers, such aspolyethyleneglycol (PEG); phospholipids; cholesterol; steroids, andpolyamines, such as PEI, spermine or spermidine.

The chemically-modified nucleotide or non-nucleotide of Formula II canbe present in one or both oligonucleotide strands of the siNA duplex,for example in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or morechemically-modified nucleotides or non-nucleotides of Formula II at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand,the antisense strand, or both strands. For example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides ornon-nucleotides of Formula II at the 5′-end of the sense strand, theantisense strand, or both strands. In another non-limiting example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modifiednucleotides or non-nucleotides of Formula II at the 3′-end of the sensestrand, the antisense strand, or both strands.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises one or more (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotideshaving Formula III:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalklylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be employed to be complementary ornon-complementary to target RNA or a non-nucleosidic base such asphenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone,pyridinone, or any other non-naturally occurring universal base that canbe complementary or non-complementary to target RNA. In one embodiment,R3 and/or R7 comprises a conjugate moiety and a linker (e.g., anucleotide or non-nucleotide linker as described herein or otherwiseknown in the art). Non-limiting examples of conjugate moieties includeligands for cellular receptors, such as peptides derived from naturallyoccurring protein ligands; protein localization sequences, includingcellular ZIP code sequences; antibodies; nucleic acid aptamers; vitaminsand other co-factors, such as folate and N-acetylgalactosamine;polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol;steroids, and polyamines, such as PEI, spermine or spermidine.

The chemically-modified nucleotide or non-nucleotide of Formula III canbe present in one or both oligonucleotide strands of the siNA duplex,for example, in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or morechemically-modified nucleotides or non-nucleotides of Formula III at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand,the antisense strand, or both strands. For example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide(s) ornon-nucleotide(s) of Formula III at the 5′-end of the sense strand, theantisense strand, or both strands. In another non-limiting example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modifiednucleotide or non-nucleotide of Formula III at the 3′-end of the sensestrand, the antisense strand, or both strands.

In another embodiment, a siNA molecule of the invention comprises anucleotide having Formula II or III, wherein the nucleotide havingFormula II or III is in an inverted configuration. For example, thenucleotide having Formula II or III is connected to the siNA constructin a 3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end,the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises a 5′-terminal phosphategroup having Formula IV:

wherein each X and Y is independently O, S, N, alkyl, substituted alkyl,or alkylhalo; wherein each Z and W is independently O, S, N, alkyl,substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, alkylhalo, oracetyl; and wherein W, X, Y and Z are not all O.

In one embodiment, the invention features a siNA molecule having a5′-terminal phosphate group having Formula IV on thetarget-complementary strand, for example, a strand complementary to atarget RNA, wherein the siNA molecule comprises an all RNA siNAmolecule. In another embodiment, the invention features a siNA moleculehaving a 5′-terminal phosphate group having Formula IV on thetarget-complementary strand wherein the siNA molecule also comprisesabout 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3′-terminalnucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or4) deoxyribonucleotides on the 3′-end of one or both strands. In anotherembodiment, a 5′-terminal phosphate group having Formula IV is presenton the target-complementary strand of a siNA molecule of the invention,for example a siNA molecule having chemical modifications having any ofFormulae I-VII.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises one or more phosphorothioateinternucleotide linkages. For example, in a non-limiting example, theinvention features a chemically-modified short interfering nucleic acid(siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioateinternucleotide linkages in one siNA strand. In yet another embodiment,the invention features a chemically-modified short interfering nucleicacid (siNA) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or morephosphorothioate internucleotide linkages in both siNA strands. Thephosphorothioate internucleotide linkages can be present in one or botholigonucleotide strands of the siNA duplex, for example in the sensestrand, the antisense strand, or both strands. The siNA molecules of theinvention can comprise one or more phosphorothioate internucleotidelinkages at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe sense strand, the antisense strand, or both strands. For example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) consecutivephosphorothioate internucleotide linkages at the 5′-end of the sensestrand, the antisense strand, or both strands. In another non-limitingexample, an exemplary siNA molecule of the invention can comprise one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidinephosphorothioate internucleotide linkages in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purinephosphorothioate internucleotide linkages in the sense strand, theantisense strand, or both strands.

In one embodiment, the invention features a siNA molecule, wherein thesense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy and/or aboutone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe sense strand; and wherein the antisense strand comprises about 1 toabout 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy,2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe antisense strand. In another embodiment, one or more, for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides ofthe sense and/or antisense siNA strand are chemically-modified with2′-deoxy, 2′-O-methyl, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or2′-deoxy-2′-fluoro nucleotides, with or without one or more, for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′-end,the 5′-end, or both of the 3′- and 5′-ends, being present in the same ordifferent strand.

In another embodiment, the invention features a siNA molecule, whereinthe sense strand comprises about 1 to about 5, specifically about 1, 2,3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1,2, 3, 4, 5, or more) universal base modified nucleotides, and optionallya terminal cap molecule at the 3-end, the 5′-end, or both of the 3′- and5′-ends of the sense strand; and wherein the antisense strand comprisesabout 1 to about 5 or more, specifically about 1, 2, 3, 4, 5, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides,and optionally a terminal cap molecule at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends of the antisense strand. In anotherembodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more, pyrimidine nucleotides of the sense and/or antisense siNAstrand are chemically-modified with 2′-deoxy, 2′-O-methyl,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy, 4′-thio and/or 2′-deoxy-2′-fluoronucleotides, with or without about 1 to about 5 or more, for exampleabout 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features a siNA molecule, wherein theantisense strand comprises one or more, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages,and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe sense strand; and wherein the antisense strand comprises about 1 toabout 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy,2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe antisense strand. In another embodiment, one or more, for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides ofthe sense and/or antisense siNA strand are chemically-modified with2′-deoxy, 2′-O-methyl, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or2′-deoxy-2′-fluoro nucleotides, with or without one or more, forexample, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′-end,the 5′-end, or both of the 3′ and 5′-ends, being present in the same ordifferent strand.

In another embodiment, the invention features a siNA molecule, whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe sense strand; and wherein the antisense strand comprises about 1 toabout 5 or more, specifically about 1, 2, 3, 4, 5 or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides,and optionally a terminal cap molecule at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends of the antisense strand. In anotherembodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more pyrimidine nucleotides of the sense and/or antisense siNA strandare chemically-modified with 2′-deoxy, 2′-O-methyl,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy, 4′-thio and/or 2′-deoxy-2′-fluoronucleotides, with or without about 1 to about 5, for example about 1, 2,3, 4, 5 or more phosphorothioate internucleotide linkages and/or aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends, being present in the same or different strand.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule having about 1 to about 5 ormore (specifically about 1, 2, 3, 4, 5 or more) phosphorothioateinternucleotide linkages in each strand of the siNA molecule.

In another embodiment, the invention features a siNA molecule comprising2′-5′ internucleotide linkages. The 2′-5′ internucleotide linkage(s) canbe at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of one orboth siNA sequence strands. In addition, the 2′-5′ internucleotidelinkage(s) can be present at various other positions within one or bothsiNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more including every internucleotide linkage of a pyrimidinenucleotide in one or both strands of the siNA molecule can comprise a2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more including every internucleotide linkage of a purine nucleotidein one or both strands of the siNA molecule can comprise a 2′-5′internucleotide linkage.

In another embodiment, a chemically-modified siNA molecule of theinvention comprises a duplex having two strands, one or both of whichcan be chemically-modified, wherein each strand is independently about15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30) nucleotides in length, wherein the duplex hasabout 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the chemicalmodification comprises a structure having any of Formulae I-VII. Forexample, an exemplary chemically-modified siNA molecule of the inventioncomprises a duplex having two strands, one or both of which can bechemically-modified with a chemical modification having any of FormulaeI-VII or any combination thereof, wherein each strand consists of about21 nucleotides, each having a 2-nucleotide 3′-terminal nucleotideoverhang, and wherein the duplex has about 19 base pairs. In anotherembodiment, a siNA molecule of the invention comprises a single strandedhairpin structure, wherein the siNA is about 36 to about 70 (e.g., about36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 15to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30) base pairs, and wherein the siNA can include achemical modification comprising a structure having any of FormulaeI-VII or any combination thereof. For example, an exemplarychemically-modified siNA molecule of the invention comprises a linearoligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45,46, 47, 48, 49, or 50) nucleotides that is chemically-modified with achemical modification having any of Formulae I-VII or any combinationthereof, wherein the linear oligonucleotide forms a hairpin structurehaving about 19 to about 21 (e.g., 19, 20, or 21) base pairs and a2-nucleotide 3′-terminal nucleotide overhang. In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.For example, a linear hairpin siNA molecule of the invention is designedsuch that degradation of the loop portion of the siNA molecule in vivocan generate a double-stranded siNA molecule with 3′-terminal overhangs,such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In another embodiment, a siNA molecule of the invention comprises ahairpin structure, wherein the siNA is about 25 to about 50 (e.g., about25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein thesiNA can include one or more chemical modifications comprising astructure having any of Formulae I-VII or any combination thereof. Forexample, an exemplary chemically-modified siNA molecule of the inventioncomprises a linear oligonucleotide having about 25 to about 35 (e.g.,about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that ischemically-modified with one or more chemical modifications having anyof Formulae I-VII or any combination thereof, wherein the linearoligonucleotide forms a hairpin structure having about 3 to about 25(e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25) base pairs and a 5′-terminal phosphategroup that can be chemically modified as described herein (for example a5′-terminal phosphate group having Formula IV). In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.In one embodiment, a linear hairpin siNA molecule of the inventioncomprises a loop portion comprising a non-nucleotide linker.

In another embodiment, a siNA molecule of the invention comprises anasymmetric hairpin structure, wherein the siNA is about 25 to about 50(e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in lengthhaving about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, andwherein the siNA can include one or more chemical modificationscomprising a structure having any of Formulae I-VII or any combinationthereof. For example, an exemplary chemically-modified siNA molecule ofthe invention comprises a linear oligonucleotide having about 25 toabout 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35)nucleotides that is chemically-modified with one or more chemicalmodifications having any of Formulae I-VII or any combination thereof,wherein the linear oligonucleotide forms an asymmetric hairpin structurehaving about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs and a5′-terminal phosphate group that can be chemically modified as describedherein (for example a 5′-terminal phosphate group having Formula IV). Inone embodiment, an asymmetric hairpin siNA molecule of the inventioncontains a stem loop motif, wherein the loop portion of the siNAmolecule is biodegradable. In another embodiment, an asymmetric hairpinsiNA molecule of the invention comprises a loop portion comprising anon-nucleotide linker.

In another embodiment, a siNA molecule of the invention comprises anasymmetric double stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length, whereinthe sense region is about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25)nucleotides in length, wherein the sense region and the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. For example, anexemplary chemically-modified siNA molecule of the invention comprisesan asymmetric double stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23)nucleotides in length and wherein the sense region is about 3 to about15 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15)nucleotides in length, wherein the sense region the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. In another embodiment,the asymmetric double stranded siNA molecule can also have a 5′-terminalphosphate group that can be chemically modified as described herein (forexample a 5′-terminal phosphate group having Formula IV).

In another embodiment, a siNA molecule of the invention comprises acircular nucleic acid molecule, wherein the siNA is about 38 to about 70(e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in lengthhaving about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the siNA caninclude a chemical modification, which comprises a structure having anyof Formulae I-VII or any combination thereof. For example, an exemplarychemically-modified siNA molecule of the invention comprises a circularoligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45,46, 47, 48, 49, or 50) nucleotides that is chemically-modified with achemical modification having any of Formulae I-VII or any combinationthereof, wherein the circular oligonucleotide forms a dumbbell shapedstructure having about 19 base pairs and 2 loops.

In another embodiment, a circular siNA molecule of the inventioncontains two loop motifs, wherein one or both loop portions of the siNAmolecule is biodegradable. For example, a circular siNA molecule of theinvention is designed such that degradation of the loop portions of thesiNA molecule in vivo can generate a double-stranded siNA molecule with3′-terminal overhangs, such as 3′-terminal nucleotide overhangscomprising about 2 nucleotides.

In one embodiment, a siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety,for example a compound having Formula V:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2. In one embodiment,R3 and/or R7 comprises a conjugate moiety and a linker (e.g., anucleotide or non-nucleotide linker as described herein or otherwiseknown in the art). Non-limiting examples of conjugate moieties includeligands for cellular receptors, such as peptides derived from naturallyoccurring protein ligands; protein localization sequences, includingcellular ZIP code sequences; antibodies; nucleic acid aptamers; vitaminsand other co-factors, such as folate and N-acetylgalactosamine;polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol;steroids, and polyamines, such as PEI, spermine or spermidine.

In one embodiment, a siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasicmoiety, for example a compound having Formula VI:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2, and either R2, R3,R8 or R13 serve as points of attachment to the siNA molecule of theinvention. In one embodiment, R3 and/or R7 comprises a conjugate moietyand a linker (e.g., a nucleotide or non-nucleotide linker as describedherein or otherwise known in the art). Non-limiting examples ofconjugate moieties include ligands for cellular receptors, such aspeptides derived from naturally occurring protein ligands; proteinlocalization sequences, including cellular ZIP code sequences;antibodies; nucleic acid aptamers; vitamins and other co-factors, suchas folate and N-acetylgalactosamine; polymers, such aspolyethyleneglycol (PEG); phospholipids; cholesterol; steroids, andpolyamines, such as PEI, spermine or spermidine.

In another embodiment, a siNA molecule of the invention comprises atleast one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)substituted polyalkyl moieties, for example a compound having FormulaVII:

wherein each n is independently an integer from 1 to 12, each R1, R2 andR3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or a group havingFormula I, and R1, R2 or R3 serves as points of attachment to the siNAmolecule of the invention. In one embodiment, R3 and/or R1 comprises aconjugate moiety and a linker (e.g., a nucleotide or non-nucleotidelinker as described herein or otherwise known in the art). Non-limitingexamples of conjugate moieties include ligands for cellular receptors,such as peptides derived from naturally occurring protein ligands;protein localization sequences, including cellular ZIP code sequences;antibodies; nucleic acid aptamers; vitamins and other co-factors, suchas folate and N-acetylgalactosamine; polymers, such aspolyethyleneglycol (PEG); phospholipids; cholesterol; steroids, andpolyamines, such as PEI, spermine or spermidine.

By “ZIP code” sequences is meant, any peptide or protein sequence thatis involved in cellular topogenic signaling mediated transport (see forexample Ray et al., 2004, Science, 306(1501): 1505)

In another embodiment, the invention features a compound having FormulaVII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises 0and is the point of attachment to the 3′-end, the 5′-end, or both of the3′ and 5′-ends of one or both strands of a double-stranded siNA moleculeof the invention or to a single-stranded siNA molecule of the invention.This modification is referred to herein as “glyceryl”.

In another embodiment, a chemically modified nucleoside ornon-nucleoside (e.g. a moiety having any of Formula V, VI or VII) of theinvention is at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends ofa siNA molecule of the invention. For example, chemically modifiednucleoside or non-nucleoside (e.g., a moiety having Formula V, VI orVII) can be present at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of the antisense strand, the sense strand, or both antisense andsense strands of the siNA molecule. In one embodiment, the chemicallymodified nucleoside or non-nucleoside (e.g., a moiety having Formula V,VI or VII) is present at the 5′-end and 3′-end of the sense strand andthe 3′-end of the antisense strand of a double stranded siNA molecule ofthe invention. In one embodiment, the chemically modified nucleoside ornon-nucleoside (e.g., a moiety having Formula V, VI or VII) is presentat the terminal position of the 5′-end and 3′-end of the sense strandand the 3′-end of the antisense strand of a double stranded siNAmolecule of the invention. In one embodiment, the chemically modifiednucleoside or non-nucleoside (e.g., a moiety having Formula V, VI orVII) is present at the two terminal positions of the 5′-end and 3′-endof the sense strand and the 3′-end of the antisense strand of a doublestranded siNA molecule of the invention. In one embodiment, thechemically modified nucleoside or non-nucleoside (e.g., a moiety havingFormula V, VI or VII) is present at the penultimate position of the5′-end and 3′-end of the sense strand and the 3′-end of the antisensestrand of a double stranded siNA molecule of the invention. In addition,a moiety having Formula VII can be present at the 3′-end or the 5′-endof a hairpin siNA molecule as described herein.

In another embodiment, a siNA molecule of the invention comprises anabasic residue having Formula V or VI, wherein the abasic residue havingFormula VI or VI is connected to the siNA construct in a3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, a siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleicacid (LNA) nucleotides, for example, at the 5′-end, the 3′-end, both ofthe 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, a siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 4′-thionucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and3′-ends, or any combination thereof, of the siNA molecule.

In another embodiment, a siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclicnucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any(e.g., one or more or all) purine nucleotides present in the senseregion are 2′-deoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-deoxy purine nucleotides or alternately a pluralityof purine nucleotides are 2′-deoxy purine nucleotides), wherein anynucleotides comprising a 3′-terminal nucleotide overhang that arepresent in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any(e.g., one or more or all) purine nucleotides present in the senseregion are 2′-O-methyl purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,or 2′-O-difluoromethoxy-ethoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), wherein any (e.g.,one or more or all) purine nucleotides present in the sense region are2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,or 2′-O-difluoromethoxy-ethoxy purine nucleotides), and wherein anynucleotides comprising a 3′-terminal nucleotide overhang that arepresent in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoro,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any(e.g., one or more or all) purine nucleotides present in the antisenseregion are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoro,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), wherein any (e.g.,one or more or all) purine nucleotides present in the antisense regionare 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-β-difluoromethoxy-ethoxy purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides), and wherein anynucleotides comprising a 3′-terminal nucleotide overhang that arepresent in said antisense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoro,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any(e.g., one or more or all) purine nucleotides present in the antisenseregion are 2′-deoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-deoxy purine nucleotides or alternately a pluralityof purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoro,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any(e.g., one or more or all) purine nucleotides present in the antisenseregion are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides (e.g., wherein all purine nucleotides are 2′-β-methyl,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention capable ofmediating RNA interference (RNAi) inside a cell or reconstituted invitro system comprising a sense region, wherein one or more pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and one or morepurine nucleotides present in the sense region are 2′-deoxy purinenucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purinenucleotides or alternately a plurality of purine nucleotides are2′-deoxy purine nucleotides), and an antisense region, wherein one ormore pyrimidine nucleotides present in the antisense region are2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidinenucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidinenucleotides or alternately a plurality of pyrimidine nucleotides are2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidinenucleotides), and one or more purine nucleotides present in theantisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides). The sense region and/orthe antisense region can have a terminal cap modification that isoptionally present at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of the sense and/or antisense sequence. The sense and/orantisense region can optionally further comprise a 3′-terminalnucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or4) 2′-deoxynucleotides. The overhang nucleotides can further compriseone or more (e.g., about 1, 2, 3, 4 or more) phosphorothioate,phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages.In any of these described embodiments, the purine nucleotides present inthe sense region are alternatively 2′-O-methyl, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,or 2′-O-difluoromethoxy-ethoxy purine nucleotides) and one or morepurine nucleotides present in the antisense region are 2′-O-methyl,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,or 2′-O-difluoromethoxy-ethoxy purine nucleotides). Also, in any ofthese embodiments, one or more purine nucleotides present in the senseregion are alternatively purine ribonucleotides (e.g., wherein allpurine nucleotides are purine ribonucleotides or alternately a pluralityof purine nucleotides are purine ribonucleotides) and any purinenucleotides present in the antisense region are 2′-O-methyl, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,or 2′-O-difluoromethoxy-ethoxy purine nucleotides). Additionally, in anyof these embodiments, one or more purine nucleotides present in thesense region and/or present in the antisense region are alternativelyselected from the group consisting of 2′-deoxy nucleotides, lockednucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides,4′-thionucleotides, 2′-O-trifluoromethyl nucleotides,2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxynucleotides and 2′-O-methyl nucleotides (e.g., wherein all purinenucleotides are selected from the group consisting of 2′-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethylnucleotides, 4′-thionucleotides, 2′-O-trifluoromethyl nucleotides,2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxynucleotides and 2′-O-methyl nucleotides or alternately a plurality ofpurine nucleotides are selected from the group consisting of 2′-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethylnucleotides, 4′-thionucleotides, 2′-O-trifluoromethyl nucleotides,2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxynucleotides and 2′-O-methyl nucleotides).

In another embodiment, any modified nucleotides present in the siNAmolecules of the invention, preferably in the antisense strand of thesiNA molecules of the invention, but also optionally in the sense and/orboth antisense and sense strands, comprise modified nucleotides havingproperties or characteristics similar to naturally occurringribonucleotides. For example, the invention features siNA moleculesincluding modified nucleotides having a Northern conformation (e.g.,Northern pseudorotation cycle, see for example Saenger, Principles ofNucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemicallymodified nucleotides present in the siNA molecules of the invention,preferably in the antisense strand of the siNA molecules of theinvention, but also optionally in the sense and/or both antisense andsense strands, are resistant to nuclease degradation while at the sametime maintaining the capacity to mediate RNAi. Non-limiting examples ofnucleotides having a northern configuration include locked nucleic acid(LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl)nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl,2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azidonucleotides, 2′-O-trifluoromethyl nucleotides,2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxynucleotides, 4′-thio nucleotides and 2′-O-methyl nucleotides.

In one embodiment, the sense strand of a double stranded siNA moleculeof the invention comprises a terminal cap moiety, such as an inverteddeoxyabaisc moiety, at the 3′-end, 5′-end, or both 3′ and 5′-ends of thesense strand.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid molecule (siNA) capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises a conjugate covalentlyattached to the chemically-modified siNA molecule. Non-limiting examplesof conjugates contemplated by the invention include conjugates andligands described in Vargeese et al., U.S. Ser. No. 10/427,160, filedApr. 30, 2003, incorporated by reference herein in its entirety,including the drawings. In another embodiment, the conjugate iscovalently attached to the chemically-modified siNA molecule via abiodegradable linker. In one embodiment, the conjugate molecule isattached at the 3′-end of either the sense strand, the antisense strand,or both strands of the chemically-modified siNA molecule. In anotherembodiment, the conjugate molecule is attached at the 5′-end of eitherthe sense strand, the antisense strand, or both strands of thechemically-modified siNA molecule. In yet another embodiment, theconjugate molecule is attached both the 3′-end and 5′-end of either thesense strand, the antisense strand, or both strands of thechemically-modified siNA molecule, or any combination thereof. In oneembodiment, a conjugate molecule of the invention comprises a moleculethat facilitates delivery of a chemically-modified siNA molecule into abiological system, such as a cell. In another embodiment, the conjugatemolecule attached to the chemically-modified siNA molecule is a ligandfor a cellular receptor, such as peptides derived from naturallyoccurring protein ligands; protein localization sequences, includingcellular ZIP code sequences; antibodies; nucleic acid aptamers; vitaminsand other co-factors, such as folate and N-acetylgalactosamine;polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol;steroids, and polyamines, such as PEI, spermine or spermidine. Examplesof specific conjugate molecules contemplated by the instant inventionthat can be attached to chemically-modified siNA molecules are describedin Vargeese et al., U.S. Ser. No. 10/201,394, filed Jul. 22, 2002incorporated by reference in its entirety herein. The type of conjugatesused and the extent of conjugation of siNA molecules of the inventioncan be evaluated for improved pharmacokinetic profiles, bioavailability,and/or stability of siNA constructs while at the same time maintainingthe ability of the siNA to mediate RNAi activity. As such, one skilledin the art can screen siNA constructs that are modified with variousconjugates to determine whether the siNA conjugate complex possessesimproved properties while maintaining the ability to mediate RNAi, forexample in animal models as are generally known in the art.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule of the invention, wherein the siNA furthercomprises a nucleotide, non-nucleotide, or mixednucleotide/non-nucleotide linker that joins the sense region of the siNAto the antisense region of the siNA. In one embodiment, a nucleotide,non-nucleotide, or mixed nucleotide/non-nucleotide linker is used, forexample, to attach a conjugate moiety to the siNA. In one embodiment, anucleotide linker of the invention can be a linker of >2 nucleotides inlength, for example about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides inlength. In another embodiment, the nucleotide linker can be a nucleicacid aptamer.

In yet another embodiment, a non-nucleotide linker of the inventioncomprises abasic nucleotide, polyether, polyamine, polyamide, peptide,carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g.polyethylene glycols such as those having between 2 and 100 ethyleneglycol units). Specific examples include those described by Seela andKaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987,15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324;Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al.,Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durandet al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides &Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301;Ono et al., Biochemistry 1991, 30:9914; Arnold et al., InternationalPublication No. WO 89/02439; Usman et al., International Publication No.WO 95/06731; Dudycz et al., International Publication No. WO 95/11910and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all herebyincorporated by reference herein. A “non-nucleotide” further means anygroup or compound that can be incorporated into a nucleic acid chain inthe place of one or more nucleotide units, including either sugar and/orphosphate substitutions, and allows the remaining bases to exhibit theirenzymatic activity. The group or compound can be abasic in that it doesnot contain a commonly recognized nucleotide base, such as adenosine,guanine, cytosine, uracil or thymine, for example at the C1 position ofthe sugar.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule capable of mediating RNA interference (RNAi) insidea cell or reconstituted in vitro system, wherein one or both strands ofthe siNA molecule that are assembled from two separate oligonucleotidesdo not comprise any ribonucleotides. For example, a siNA molecule can beassembled from a single oligonucleotide where the sense and antisenseregions of the siNA comprise separate oligonucleotides that do not haveany ribonucleotides (e.g., nucleotides having a 2′-OH group) present inthe oligonucleotides. In another example, a siNA molecule can beassembled from a single oligonucleotide where the sense and antisenseregions of the siNA are linked or circularized by a nucleotide ornon-nucleotide linker as described herein, wherein the oligonucleotidedoes not have any ribonucleotides (e.g., nucleotides having a 2′-OHgroup) present in the oligonucleotide. Applicant has surprisingly foundthat the presence of ribonucleotides (e.g., nucleotides having a2′-hydroxyl group) within the siNA molecule is not required or essentialto support RNAi activity. As such, in one embodiment, all positionswithin the siNA can include chemically modified nucleotides and/ornon-nucleotides such as nucleotides and or non-nucleotides havingFormula I, II, III, IV, V, VI, or VII or any combination thereof to theextent that the ability of the siNA molecule to support RNAi activity ina cell is maintained.

In one embodiment, a siNA molecule of the invention is a single strandedsiNA molecule that mediates RNAi activity in a cell or reconstituted invitro system comprising a single stranded polynucleotide havingcomplementarity to a target nucleic acid sequence. In anotherembodiment, the single stranded siNA molecule of the invention comprisesa 5′-terminal phosphate group. In another embodiment, the singlestranded siNA molecule of the invention comprises a 5′-terminalphosphate group and a 3′-terminal phosphate group (e.g., a 2′,3′-cyclicphosphate). In another embodiment, the single stranded siNA molecule ofthe invention comprises about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. Inyet another embodiment, the single stranded siNA molecule of theinvention comprises one or more chemically modified nucleotides ornon-nucleotides described herein. For example, all the positions withinthe siNA molecule can include chemically-modified nucleotides such asnucleotides having any of Formulae I-VII, or any combination thereof tothe extent that the ability of the siNA molecule to support RNAiactivity in a cell is maintained.

In one embodiment, a siNA molecule of the invention is a single strandedsiNA molecule that mediates RNAi activity in a cell or reconstituted invitro system comprising a single stranded polynucleotide havingcomplementarity to a target nucleic acid sequence, wherein one or morepyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein anypurine nucleotides present in the antisense region are 2′-O-methyl,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,or 2′-O-difluoromethoxy-ethoxy purine nucleotides), and a terminal capmodification that is optionally present at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the antisense sequence. The siNAoptionally further comprises about 1 to about 4 or more (e.g., about 1,2, 3, 4 or more) terminal 2′-deoxynucleotides at the 3′-end of the siNAmolecule, wherein the terminal nucleotides can further comprise one ormore (e.g., 1, 2, 3, 4 or more) phosphorothioate, phosphonoacetate,and/or thiophosphonoacetate internucleotide linkages, and wherein thesiNA optionally further comprises a terminal phosphate group, such as a5′-terminal phosphate group. In any of these embodiments, any purinenucleotides present in the antisense region are alternatively 2′-deoxypurine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxypurine nucleotides or alternately a plurality of purine nucleotides are2′-deoxy purine nucleotides). Also, in any of these embodiments, anypurine nucleotides present in the siNA (i.e., purine nucleotides presentin the sense and/or antisense region) can alternatively be lockednucleic acid (LNA) nucleotides (e.g., wherein all purine nucleotides areLNA nucleotides or alternately a plurality of purine nucleotides are LNAnucleotides). Also, in any of these embodiments, any purine nucleotidespresent in the siNA are alternatively 2′-methoxyethyl purine nucleotides(e.g., wherein all purine nucleotides are 2′-methoxyethyl purinenucleotides or alternately a plurality of purine nucleotides are2′-methoxyethyl purine nucleotides). In another embodiment, any modifiednucleotides present in the single stranded siNA molecules of theinvention comprise modified nucleotides having properties orcharacteristics similar to naturally occurring ribonucleotides. Forexample, the invention features siNA molecules including modifiednucleotides having a Northern conformation (e.g., Northernpseudorotation cycle, see for example Saenger, Principles of NucleicAcid Structure, Springer-Verlag ed., 1984). As such, chemically modifiednucleotides present in the single stranded siNA molecules of theinvention are preferably resistant to nuclease degradation while at thesame time maintaining the capacity to mediate RNAi.

In one embodiment, a siNA molecule of the invention comprises chemicallymodified nucleotides or non-nucleotides (e.g., having any of FormulaeI-VII, such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides) at alternatingpositions within one or more strands or regions of the siNA molecule.For example, such chemical modifications can be introduced at everyother position of a RNA based siNA molecule, starting at either thefirst or second nucleotide from the 3′-end or 5′-end of the siNA. In anon-limiting example, a double stranded siNA molecule of the inventionin which each strand of the siNA is 21 nucleotides in length is featuredwherein positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 of eachstrand are chemically modified (e.g., with compounds having any ofFormulae I-VII, such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides). In anothernon-limiting example, a double stranded siNA molecule of the inventionin which each strand of the siNA is 21 nucleotides in length is featuredwherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of each strandare chemically modified (e.g., with compounds having any of FormulaeI-VII, such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides). Such siNAmolecules can further comprise terminal cap moieties and/or backbonemodifications as described herein.

In one embodiment, the invention features a method for delivering oradministering a biologically active molecule, such as a polynucleotidemolecule (e.g., siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleic acidmolecule) of the invention to a cell or cells in a subject or organism,comprising administering a formulation or composition of the inventionunder conditions suitable for delivery of the polynucleotide componentof the formulation or composition to the cell or cells of the subject ororganism. In separate embodiments, the cell is, for example, a lungcell, liver cell, CNS cell, PNS cell, tumor cell, kidney cell, vascularcell, skin cell, ocular cell, or cells of the ear.

In one embodiment, the invention features a method for delivering oradministering a biologically active molecule, such as a polynucleotidemolecule (e.g., siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleic acidmolecule) of the invention to liver or liver cells (e.g., hepatocytes)in a subject or organism, comprising administering a formulation orcomposition of the invention under conditions suitable for delivery ofthe polynucleotide component of the formulation or composition to theliver or liver cells (e.g., hepatocytes) of the subject or organism.

In one embodiment, the invention features a method for modulating theexpression of a target gene within a cell comprising, introducing aformulation or composition of the invention into a cell under conditionssuitable to modulate the expression of the target gene in the cell. Inone embodiment, the cell is a liver cell (e.g., hepatocyte). In otherembodiments, the cell is, for example, a lung cell, CNS cell, PNS cell,tumor cell, kidney cell, vascular cell, skin cell, ocular cell, or cellsof the ear. In one embodiment, the formulation or composition comprisesa polynucleotide, such as a siNA, miRNA, RNAi inhibitor, antisense,aptamer, decoy, ribozyme, 2-5A, triplex forming oligonucleotide, orother nucleic acid molecule.

In another embodiment, the invention features a method for modulatingthe expression of more than one target gene within a cell comprising,introducing a formulation or composition of the invention into the cellunder conditions suitable to modulate the expression of the target genesin the cell. In one embodiment, the cell is a liver cell (e.g.,hepatocyte). In other embodiments, the cell is, for example, a lungcell, CNS cell, PNS cell, tumor cell, kidney cell, vascular cell, skincell, ocular cell, or cells of the ear. In one embodiment, theformulation or composition comprises a polynucleotide, such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule.

In one embodiment, the invention features a method for treating orpreventing a disease, disorder, trait or condition related to geneexpression in a subject or organism comprising contacting the subject ororganism with a formulation or composition of the invention underconditions suitable to modulate the expression of the target gene in thesubject or organism. In one embodiment, the formulation or compositioncomprises a polynucleotide, such as a siNA, miRNA, RNAi inhibitor,antisense, aptamer, decoy, ribozyme, 2-5A, triplex formingoligonucleotide, or other nucleic acid molecule. In one embodiment, thereduction of gene expression and thus reduction in the level of therespective protein/RNA relieves, to some extent, the symptoms of thedisease, disorder, trait or condition.

In one embodiment, the invention features a method for treating orpreventing cancer in a subject or organism comprising contacting thesubject or organism with a formulation or composition of the inventionunder conditions suitable to modulate the expression of the target genein the subject or organism whereby the treatment or prevention of cancercan be achieved. In one embodiment, the formulation or compositioncomprises a polynucleotide, such as a siNA, miRNA, RNAi inhibitor,antisense, aptamer, decoy, ribozyme, 2-5A, triplex formingoligonucleotide, or other nucleic acid molecule. In one embodiment, theinvention features contacting the subject or organism with a formulationor composition of the invention via local administration to relevanttissues or cells, such as cancerous cells and tissues. In oneembodiment, the invention features contacting the subject or organismwith a formulation or composition of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof the formulation or composition) to relevant tissues or cells, such astissues or cells involved in the maintenance or development of cancer ina subject or organism. The formulation or composition of the inventioncan be formulated or conjugated as described herein or otherwise knownin the art to target appropriate tissues or cells in the subject ororganism.

In one embodiment, the invention features a method for treating orpreventing a proliferative disease or condition in a subject or organismcomprising contacting the subject or organism with a formulation orcomposition of the invention under conditions suitable to modulate theexpression of the target gene in the subject or organism whereby thetreatment or prevention of the proliferative disease or condition can beachieved. In one embodiment, the formulation or composition comprises apolynucleotide, such as a siNA, miRNA, RNAi inhibitor, antisense,aptamer, decoy, ribozyme, 2-5A, triplex forming oligonucleotide, orother nucleic acid molecule. In one embodiment, the invention featurescontacting the subject or organism with a formulation or composition ofthe invention via local administration to relevant tissues or cells,such as cells and tissues involved in proliferative disease. In oneembodiment, the invention features contacting the subject or organismwith a formulation or composition of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof the formulation or composition) to relevant tissues or cells, such astissues or cells involved in the maintenance or development of theproliferative disease or condition in a subject or organism. Theformulation or composition of the invention can be formulated orconjugated as described herein or otherwise known in the art to targetappropriate tissues or cells in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing transplant and/or tissue rejection (allograft rejection) in asubject or organism comprising contacting the subject or organism with aformulation or composition of the invention under conditions suitable tomodulate the expression of the target gene in the subject or organismwhereby the treatment or prevention of transplant and/or tissuerejection (allograft rejection) can be achieved. In one embodiment, theformulation or composition comprises a polynucleotide, such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule. In oneembodiment, the invention features contacting the subject or organismwith a formulation or composition of the invention via localadministration to relevant tissues or cells, such as cells and tissuesinvolved in transplant and/or tissue rejection (allograft rejection). Inone embodiment, the invention features contacting the subject ororganism with a formulation or composition of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof the formulation or composition) to relevant tissues or cells, such astissues or cells involved in the maintenance or development oftransplant and/or tissue rejection (allograft rejection) in a subject ororganism. The formulation or composition of the invention can beformulated or conjugated as described herein or otherwise known in theart to target appropriate tissues or cells in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing an autoimmune disease, disorder, trait or condition in asubject or organism comprising contacting the subject or organism with aformulation or composition of the invention under conditions suitable tomodulate the expression of the target gene in the subject or organismwhereby the treatment or prevention of the autoimmune disease, disorder,trait or condition can be achieved. In one embodiment, the formulationor composition comprises a polynucleotide, such as a siNA, miRNA, RNAiinhibitor, antisense, aptamer, decoy, ribozyme, 2-5A, triplex formingoligonucleotide, or other nucleic acid molecule. In one embodiment, theinvention features contacting the subject or organism with a formulationor composition of the invention via local administration to relevanttissues or cells, such as cells and tissues involved in the autoimmunedisease, disorder, trait or condition. In one embodiment, the inventionfeatures contacting the subject or organism with a formulation orcomposition of the invention via systemic administration (such as viaintravenous or subcutaneous administration of the formulation orcomposition) to relevant tissues or cells, such as tissues or cellsinvolved in the maintenance or development of the autoimmune disease,disorder, trait or condition in a subject or organism. The formulationor composition of the invention can be formulated or conjugated asdescribed herein or otherwise known in the art to target appropriatetissues or cells in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing an infectious disease, disorder, trait or condition in asubject or organism comprising contacting the subject or organism with aformulation or composition of the invention under conditions suitable tomodulate the expression of the target gene in the subject or organismwhereby the treatment or prevention of the infectious disease, disorder,trait or condition can be achieved. In one embodiment, the formulationor composition comprises a polynucleotide, such as a siNA, miRNA, RNAiinhibitor, antisense, aptamer, decoy, ribozyme, 2-5A, triplex formingoligonucleotide, or other nucleic acid molecule. In one embodiment, theinvention features contacting the subject or organism with a formulationor composition of the invention via local administration to relevanttissues or cells, such as cells and tissues involved in the infectiousdisease, disorder, trait or condition. In one embodiment, the inventionfeatures contacting the subject or organism with a formulation orcomposition of the invention via systemic administration (such as viaintravenous or subcutaneous administration of the formulation orcomposition) to relevant tissues or cells, such as tissues or cellsinvolved in the maintenance or development of the infectious disease,disorder, trait or condition in a subject or organism. The formulationor composition of the invention can be formulated or conjugated asdescribed herein or otherwise known in the art to target appropriatetissues or cells in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing an age-related disease, disorder, trait or condition in asubject or organism comprising contacting the subject or organism with aformulation or composition of the invention under conditions suitable tomodulate the expression of the target gene in the subject or organismwhereby the treatment or prevention of the age-related disease,disorder, trait or condition can be achieved. In one embodiment, theformulation or composition comprises a polynucleotide, such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule. In oneembodiment, the invention features contacting the subject or organismwith a formulation or composition of the invention via localadministration to relevant tissues or cells, such as cells and tissuesinvolved in the age-related disease, disorder, trait or condition. Inone embodiment, the invention features contacting the subject ororganism with a formulation or composition of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof the formulation or composition) to relevant tissues or cells, such astissues or cells involved in the maintenance or development of theage-related disease, disorder, trait or condition in a subject ororganism. The formulation or composition of the invention can beformulated or conjugated as described herein or otherwise known in theart to target appropriate tissues or cells in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing a neurologic or neurodegenerative disease, disorder, trait orcondition in a subject or organism comprising contacting the subject ororganism with a formulation or composition of the invention underconditions suitable to modulate the expression of the target gene in thesubject or organism whereby the treatment or prevention of theneurologic or neurodegenerative disease, disorder, trait or conditioncan be achieved. In one embodiment, the formulation or compositioncomprises a polynucleotide, such as a siNA, miRNA, RNAi inhibitor,antisense, aptamer, decoy, ribozyme, 2-5A, triplex formingoligonucleotide, or other nucleic acid molecule. In one embodiment, theinvention features contacting the subject or organism with a formulationor composition of the invention via local administration to relevanttissues or cells, such as cells and tissues involved in the neurologicor neurodegenerative disease, disorder, trait or condition. In oneembodiment, the invention features contacting the subject or organismwith a formulation or composition of the invention via systemicadministration (such as via catheterization, osmotic pump administration(e.g., intrathecal or ventricular) intravenous or subcutaneousadministration of the formulation or composition) to relevant tissues orcells, such as tissues or cells involved in the maintenance ordevelopment of the neurologic or neurodegenerative disease, disorder,trait or condition in a subject or organism. The formulation orcomposition of the invention can be formulated or conjugated asdescribed herein or otherwise known in the art to target appropriatetissues or cells in the subject or organism. In one embodiment, theneurologic disease is Huntington disease.

In one embodiment, the invention features a method for treating orpreventing a metabolic disease, disorder, trait or condition in asubject or organism comprising contacting the subject or organism with aformulation or composition of the invention under conditions suitable tomodulate the expression of the target gene in the subject or organismwhereby the treatment or prevention of the metabolic disease, disorder,trait or condition can be achieved. In one embodiment, the formulationor composition comprises a polynucleotide, such as a siNA, miRNA, RNAiinhibitor, antisense, aptamer, decoy, ribozyme, 2-5A, triplex formingoligonucleotide, or other nucleic acid molecule. In one embodiment, theinvention features contacting the subject or organism with a formulationor composition of the invention via local administration to relevanttissues or cells, such as cells and tissues involved in the metabolicdisease, disorder, trait or condition. In one embodiment, the inventionfeatures contacting the subject or organism with a formulation orcomposition of the invention via systemic administration (such as viaintravenous or subcutaneous administration of the formulation orcomposition) to relevant tissues or cells, such as tissues or cellsinvolved in the maintenance or development of the metabolic disease,disorder, trait or condition in a subject or organism. The formulationor composition of the invention can be formulated or conjugated asdescribed herein or otherwise known in the art to target appropriatetissues or cells in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing a cardiovascular disease, disorder, trait or condition in asubject or organism comprising contacting the subject or organism with aformulation or composition of the invention under conditions suitable tomodulate the expression of the target gene in the subject or organismwhereby the treatment or prevention of the cardiovascular disease,disorder, trait or condition can be achieved. In one embodiment, theformulation or composition comprises a polynucleotide, such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule. In oneembodiment, the invention features contacting the subject or organismwith a formulation or composition of the invention via localadministration to relevant tissues or cells, such as cells and tissuesinvolved in the cardiovascular disease, disorder, trait or condition. Inone embodiment, the invention features contacting the subject ororganism with a formulation or composition of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof the formulation or composition) to relevant tissues or cells, such astissues or cells involved in the maintenance or development of thecardiovascular disease, disorder, trait or condition in a subject ororganism. The formulation or composition of the invention can beformulated or conjugated as described herein or otherwise known in theart to target appropriate tissues or cells in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing a respiratory disease, disorder, trait or condition in asubject or organism comprising contacting the subject or organism with aformulation or composition of the invention under conditions suitable tomodulate the expression of the target gene in the subject or organismwhereby the treatment or prevention of the respiratory disease,disorder, trait or condition can be achieved. In one embodiment, theformulation or composition comprises a polynucleotide, such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule. In oneembodiment, the invention features contacting the subject or organismwith a formulation or composition of the invention via localadministration to relevant tissues or cells, such as cells and tissuesinvolved in the respiratory disease, disorder, trait or condition. Inone embodiment, the invention features contacting the subject ororganism with a formulation or composition of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof the formulation or composition) to relevant tissues or cells, such astissues or cells involved in the maintenance or development of therespiratory disease, disorder, trait or condition in a subject ororganism. The formulation or composition of the invention can beformulated or conjugated as described herein or otherwise known in theart to target appropriate tissues or cells in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing an ocular disease, disorder, trait or condition in a subjector organism comprising contacting the subject or organism with aformulation or composition of the invention under conditions suitable tomodulate the expression of the target gene in the subject or organismwhereby the treatment or prevention of the ocular disease, disorder,trait or condition can be achieved. In one embodiment, the formulationor composition comprises a polynucleotide, such as a siNA, miRNA, RNAiinhibitor, antisense, aptamer, decoy, ribozyme, 2-5A, triplex formingoligonucleotide, or other nucleic acid molecule. In one embodiment, theinvention features contacting the subject or organism with a formulationor composition of the invention via local administration to relevanttissues or cells, such as cells and tissues involved in the oculardisease, disorder, trait or condition. In one embodiment, the inventionfeatures contacting the subject or organism with a formulation orcomposition of the invention via systemic administration (such as viaintravenous or subcutaneous administration of the formulation orcomposition) to relevant tissues or cells, such as tissues or cellsinvolved in the maintenance or development of the ocular disease,disorder, trait or condition in a subject or organism. The formulationor composition of the invention can be formulated or conjugated asdescribed herein or otherwise known in the art to target appropriatetissues or cells in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing a dermatological disease, disorder, trait or condition in asubject or organism comprising contacting the subject or organism with aformulation or composition of the invention under conditions suitable tomodulate the expression of the target gene in the subject or organismwhereby the treatment or prevention of the dermatological disease,disorder, trait or condition can be achieved. In one embodiment, theformulation or composition comprises a polynucleotide, such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule. In oneembodiment, the invention features contacting the subject or organismwith a formulation or composition of the invention via localadministration to relevant tissues or cells, such as cells and tissuesinvolved in the dermatological disease, disorder, trait or condition. Inone embodiment, the invention features contacting the subject ororganism with a formulation or composition of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof the formulation or composition) to relevant tissues or cells, such astissues or cells involved in the maintenance or development of thedermatological disease, disorder, trait or condition in a subject ororganism. The formulation or composition of the invention can beformulated or conjugated as described herein or otherwise known in theart to target appropriate tissues or cells in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing a liver disease, disorder, trait or condition (e.g.,hepatitis, HCV, HBV, diabetes, cirrhosis, hepatocellular carcinoma etc.)in a subject or organism comprising contacting the subject or organismwith a formulation or composition of the invention under conditionssuitable to modulate the expression of the target gene in the subject ororganism whereby the treatment or prevention of the liver disease,disorder, trait or condition can be achieved. In one embodiment, theformulation or composition comprises a polynucleotide, such as a siNA,miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule. In oneembodiment, the invention features contacting the subject or organismwith a formulation or composition of the invention via localadministration to relevant tissues or cells, such as liver cells andtissues involved in the liver disease, disorder, trait or condition. Inone embodiment, the invention features contacting the subject ororganism with a formulation or composition of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof the formulation or composition) to relevant tissues or cells, such astissues or cells involved in the maintenance or development of the liverdisease, disorder, trait or condition in a subject or organism. Theformulation or composition of the invention can be formulated orconjugated as described herein or otherwise known in the art to targetappropriate tissues or cells in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing a kidney/renal disease, disorder, trait or condition (e.g.,polycystic kidney disease etc.) in a subject or organism comprisingcontacting the subject or organism with a formulation or composition ofthe invention under conditions suitable to modulate the expression ofthe target gene in the subject or organism whereby the treatment orprevention of the kidney/renal disease, disorder, trait or condition canbe achieved. In one embodiment, the formulation or composition comprisesa polynucleotide, such as a siNA, miRNA, RNAi inhibitor, antisense,aptamer, decoy, ribozyme, 2-5A, triplex forming oligonucleotide, orother nucleic acid molecule. In one embodiment, the invention featurescontacting the subject or organism with a formulation or composition ofthe invention via local administration to relevant tissues or cells,such as kidney/renal cells and tissues involved in the kidney/renaldisease, disorder, trait or condition. In one embodiment, the inventionfeatures contacting the subject or organism with a formulation orcomposition of the invention via systemic administration (such as viaintravenous or subcutaneous administration of the formulation orcomposition) to relevant tissues or cells, such as tissues or cellsinvolved in the maintenance or development of the kidney/renal disease,disorder, trait or condition in a subject or organism. The formulationor composition of the invention can be formulated or conjugated asdescribed herein or otherwise known in the art to target appropriatetissues or cells in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing an auditory disease, disorder, trait or condition (e.g.,hearing loss, deafness, etc.) in a subject or organism comprisingcontacting the subject or organism with a formulation or composition ofthe invention under conditions suitable to modulate the expression ofthe target gene in the subject or organism whereby the treatment orprevention of the auditory disease, disorder, trait or condition can beachieved. In one embodiment, the formulation or composition comprises apolynucleotide, such as a siNA, miRNA, RNAi inhibitor, antisense,aptamer, decoy, ribozyme, 2-5A, triplex forming oligonucleotide, orother nucleic acid molecule. In one embodiment, the invention featurescontacting the subject or organism with a formulation or composition ofthe invention via local administration to relevant tissues or cells,such as cells and tissues of the ear, inner hear, or middle ear involvedin the auditory disease, disorder, trait or condition. In oneembodiment, the invention features contacting the subject or organismwith a formulation or composition of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof the formulation or composition) to relevant tissues or cells, such astissues or cells involved in the maintenance or development of theauditory disease, disorder, trait or condition in a subject or organism.The formulation or composition of the invention can be formulated orconjugated as described herein or otherwise known in the art to targetappropriate tissues or cells in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing a disease or condition as described herein in a subject ororganism, comprising administering to the subject or organism aformulation or composition of the invention; wherein the formulation orcomposition is administered under conditions suitable for reducing orinhibiting the level of target gene expression in the subject comparedto a subject not treated with the formulation or composition. In oneembodiment, the formulation or composition comprises a lipidnanoparticle and a siNA molecule of the invention.

In one embodiment, the invention features a method for treating orpreventing a disease or condition as described herein in a subject ororganism, comprising administering to the subject a formulation orcomposition of the invention; wherein (a) the formulated molecularcomposition comprises a double stranded nucleic acid molecule having asense strand and an antisense strand; (b) each strand of the doublestranded nucleic acid molecule is 15 to 28 nucleotides in length; (c) atleast 15 nucleotides of the sense strand are complementary to theantisense strand (d) the antisense strand of the double stranded nucleicacid molecule has complementarity to a target RNA; and wherein theformulation or composition is administered under conditions suitable forreducing or inhibiting the target RNA in the subject compared to asubject not treated with the formulation or composition. In oneembodiment, the formulation or composition comprises a lipidnanoparticle and a siNA molecule of the invention.

In one embodiment, the invention features a method for treating orpreventing a disease or condition as described herein in a subject ororganism, comprising administering to the subject a formulation orcomposition of the invention; wherein (a) the formulated molecularcomposition comprises a double stranded nucleic acid molecule having asense strand and an antisense strand; (b) each strand of the doublestranded nucleic acid molecule is 15 to 28 nucleotides in length; (c) atleast 15 nucleotides of the sense strand are complementary to theantisense strand (d) the antisense strand of the double stranded nucleicacid molecule has complementarity to a target RNA; (e) at least 20% ofthe internal nucleotides of each strand of the double stranded nucleicacid molecule are modified nucleosides having a chemical modification;and (f) at least two of the chemical modifications are different fromeach other, and wherein the formulation or composition is administeredunder conditions suitable for reducing or inhibiting the level of targetRNA in the subject compared to a subject not treated with theformulation or composition. In one embodiment, the formulation orcomposition comprises a lipid nanoparticle and a siNA molecule of theinvention.

In any of the methods of treatment of the invention, the formulation orcomposition can be administered to the subject as a course of treatment,for example administration at various time intervals, such as once perday over the course of treatment, once every two days over the course oftreatment, once every three days over the course of treatment, onceevery four days over the course of treatment, once every five days overthe course of treatment, once every six days over the course oftreatment, once per week over the course of treatment, once every otherweek over the course of treatment, once per month over the course oftreatment, etc. In one embodiment, the course of treatment is once every1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In one embodiment, the course oftreatment is from about one to about 52 weeks or longer (e.g.,indefinitely). In one embodiment, the course of treatment is from aboutone to about 48 months or longer (e.g., indefinitely).

In one embodiment, a course of treatment involves an initial course oftreatment, such as once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreweeks for a fixed interval (e.g., 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×,10× or more) followed by a maintenance course of treatment, such as onceevery 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, or more weeks for anadditional fixed interval (e.g., 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×or more).

In any of the methods of treatment of the invention, the formulation orcomposition can be administered to the subject systemically as describedherein or otherwise known in the art. Systemic administration caninclude, for example, intravenous, subcutaneous, intramuscular,catheterization, nasopharangeal, transdermal, or gastrointestinaladministration as is generally known in the art.

In one embodiment, in any of the methods of treatment or prevention ofthe invention, the formulation or composition can be administered to thesubject locally or to local tissues as described herein or otherwiseknown in the art. Local administration can include, for example,catheterization, implantation, osmotic pumping, direct injection,dermal/transdermal application, stenting, ear/eye drops, or portal veinadministration to relevant tissues, or any other local administrationtechnique, method or procedure, as is generally known in the art.

In one embodiment, the invention features a composition comprising aformulation or composition of the invention, in a pharmaceuticallyacceptable carrier or diluent. In another embodiment, the inventionfeatures a pharmaceutical composition comprising formulation orcompositions of the invention, targeting one or more genes in apharmaceutically acceptable carrier or diluent. In another embodiment,the invention features a method for diagnosing a disease or condition ina subject comprising administering to the subject a formulation orcomposition of the invention under conditions suitable for the diagnosisof the disease or condition in the subject. In another embodiment, theinvention features a method for treating or preventing a disease, trait,or condition in a subject, comprising administering to the subject aformulation or composition of the invention under conditions suitablefor the treatment or prevention of the disease, trait or condition inthe subject, alone or in conjunction with one or more other therapeuticcompounds.

In one embodiment, the method of synthesis of polynucleotide moleculesof the invention, including but not limited to siNA, miRNA, RNAiinhibitor, antisense, aptamer, decoy, ribozyme, 2-5A, triplex formingoligonucleotide, or other nucleic acid molecules, comprises theteachings of Scaringe et al., U.S. Pat. Nos. 5,889,136; 6,008,400; and6,111,086, incorporated by reference herein in their entirety.

In another embodiment, the invention features a method for generatingformulated polynucleotide (e.g., to siNA, miRNA, RNAi inhibitor,antisense, aptamer, decoy, ribozyme, 2-5A, triplex formingoligonucleotide, or other nucleic acid molecule) compositions withincreased nuclease resistance comprising (a) introducing modifiednucleotides into a polynucleotide component of a formulation orcomposition of the invention, and (b) assaying the formulation orcomposition of step (a) under conditions suitable for isolatingformulated polynucleotide compositions having increased nucleaseresistance.

In another embodiment, the invention features a method for generatingpolynucleotide (e.g., to siNA, miRNA, RNAi inhibitor, antisense,aptamer, decoy, ribozyme, 2-5A, triplex forming oligonucleotide, orother nucleic acid molecule) molecules with improved toxicologicprofiles (e.g., having attenuated or no immunstimulatory properties)comprising (a) introducing nucleotides having any of Formula I-VII(e.g., siNA motifs referred to in Table I) or any combination thereofinto a polynucleotide molecule, and (b) assaying the polynucleotidemolecule of step (a) under conditions suitable for isolating siNAmolecules having improved toxicologic profiles.

In another embodiment, the invention features a method for generatingformulated siNA compositions with improved toxicologic profiles (e.g.,having attenuated or no immunstimulatory properties) comprising (a)generating a formulated siNA composition comprising a siNA molecule ofthe invention and a delivery vehicle or delivery particle as describedherein or as otherwise known in the art, and (b) assaying the siNAformulation of step (a) under conditions suitable for isolatingformulated siNA compositions having improved toxicologic profiles.

In another embodiment, the invention features a method for generatingsiNA molecules that do not stimulate an interferon response (e.g., nointerferon response or attenuated interferon response) in a cell,subject, or organism, comprising (a) introducing nucleotides having anyof Formula I-VII (e.g., siNA motifs referred to in Table I) or anycombination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules that do not stimulate an interferon response.

In another embodiment, the invention features a method for generatingformulated siNA compositions that do not stimulate an interferonresponse (e.g., no interferon response or attenuated interferonresponse) in a cell, subject, or organism, comprising (a) generating aformulated siNA composition comprising a siNA molecule of the inventionand a delivery vehicle or delivery particle as described herein or asotherwise known in the art, and (b) assaying the siNA formulation ofstep (a) under conditions suitable for isolating formulated siNAcompositions that do not stimulate an interferon response. In oneembodiment, the interferon comprises interferon alpha.

In another embodiment, the invention features a method for generatingsiNA molecules that do not stimulate an inflammatory or proinflammatorycytokine response (e.g., no cytokine response or attenuated cytokineresponse) in a cell, subject, or organism, comprising (a) introducingnucleotides having any of Formula I-VII (e.g., siNA motifs referred toin Table I) or any combination thereof into a siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules that do not stimulate a cytokine response. Inone embodiment, the cytokine comprises an interleukin such asinterleukin-6 (IL-6) and/or tumor necrosis factor alpha (TNF-α).

In another embodiment, the invention features a method for generatingformulated siNA compositions that do not stimulate an inflammatory orproinflammatory cytokine response (e.g., no cytokine response orattenuated cytokine response) in a cell, subject, or organism,comprising (a) generating a formulated siNA composition comprising asiNA molecule of the invention and a delivery vehicle or deliveryparticle as described herein or as otherwise known in the art, and (b)assaying the siNA formulation of step (a) under conditions suitable forisolating formulated siNA compositions that do not stimulate a cytokineresponse. In one embodiment, the cytokine comprises an interleukin suchas interleukin-6 (IL-6) and/or tumor necrosis alpha (TNF-α).

In another embodiment, the invention features a method for generatingsiNA molecules that do not stimulate Toll-like Receptor (TLR) response(e.g., no TLR response or attenuated TLR response) in a cell, subject,or organism, comprising (a) introducing nucleotides having any ofFormula I-VII (e.g., siNA motifs referred to in Table I) or anycombination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules that do not stimulate a TLR response. In one embodiment, theTLR comprises TLR3, TLR7, TLR8 and/or TLR9.

In another embodiment, the invention features a method for generatingformulated siNA compositions that do not stimulate a Toll-like Receptor(TLR) response (e.g., no TLR response or attenuated TLR response) in acell, subject, or organism, comprising (a) generating a formulated siNAcomposition comprising a siNA molecule of the invention and a deliveryvehicle or delivery particle as described herein or as otherwise knownin the art, and (b) assaying the siNA formulation of step (a) underconditions suitable for isolating formulated siNA compositions that donot stimulate a TLR response. In one embodiment, the TLR comprises TLR3,TLR7, TLR8 and/or TLR9.

By “improved toxicologic profile”, is meant that the polynucleotide,formulation or composition, siNA or formulated siNA composition exhibitsdecreased toxicity in a cell, subject, or organism compared to anunmodified polynucleotide, formulation or composition, siNA orformulated siNA composition, or siNA molecule having fewer modificationsor modifications that are less effective in imparting improvedtoxicology. In a non-limiting example, polynucleotides, formulation orcomposition s, siNAs or formulated siNA compositions with improvedtoxicologic profiles are associated with reduced immunostimulatoryproperties, such as a reduced, decreased or attenuated immunostimulatoryresponse in a cell, subject, or organism compared to an unmodifiedpolynucleotide, formulation or composition, siNA or formulated siNAcomposition, or polynucleotide (e.g., siNA) molecule having fewermodifications or modifications that are less effective in impartingimproved toxicology. Such an improved toxicologic profile ischaracterized by abrogated or reduced immunostimulation, such asreduction or abrogation of induction of interferons (e.g., interferonalpha), inflammatory cytokines (e.g., interleukins such as IL-6, and/orTNF-alpha), and/or toll like receptors (e.g., TLR-3, TLR-7, TLR-8,and/or TLR-9). In one embodiment, a polynucleotide, formulation orcomposition, siNA or formulated siNA composition with an improvedtoxicological profile comprises no ribonucleotides. In one embodiment, apolynucleotide, formulation or composition, siNA or formulated siNAcomposition with an improved toxicological profile comprises less than 5ribonucleotides (e.g., 1, 2, 3, or 4 ribonucleotides). In oneembodiment, a siNA or formulated siNA composition with an improvedtoxicological profile comprises Stab 7, Stab 8, Stab 11, Stab 12, Stab13, Stab 16, Stab 17, Stab 18, Stab 19, Stab 20, Stab 23, Stab 24, Stab25, Stab 26, Stab 27, Stab 28, Stab 29, Stab 30, Stab 31, Stab 32, Stab33, Stab 34 or any combination thereof (see Table I). Herein, numericStab chemistries include both 2′-fluoro and 2′-OCF3 versions of thechemistries shown in Table I. For example, “Stab ⅞” refers to both Stab7/8 and Stab 7F/8F etc. In one embodiment, a siNA or formulated siNAcomposition with an improved toxicological profile comprises a siNAmolecule as described in United States Patent Application PublicationNo. 20030077829, incorporated by reference herein in its entiretyincluding the drawings.

In one embodiment, the level of immunostimulatory response associatedwith a given polynucleotide, formulation or composition, siNA moleculeor formulated siNA composition can be measured as is described herein oras is otherwise known in the art, for example by determining the levelof PKR/interferon response, proliferation, B-cell activation, and/orcytokine production in assays to quantitate the immunostimulatoryresponse of particular polynucleotide molecules (see, for example,Leifer et al., 2003, J Immunother. 26, 313-9; and U.S. Pat. No.5,968,909, incorporated in its entirety by reference). In oneembodiment, the reduced immunostimulatory response is between about 10%and about 100% compared to an unmodified or minimally modified siRNAmolecule, e.g., about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or 100%reduced immunostimulatory response. In one embodiment, theimmunostimulatory response associated with a siNA molecule can bemodulated by the degree of chemical modification. For example, a siNAmolecule having between about 10% and about 100%, e.g., about 10%,20%,30%,40%, 50%, 60%, 70%, 80%, 90% or 100% of the nucleotide positionsin the siNA molecule modified can be selected to have a correspondingdegree of immunostimulatory properties as described herein.

In one embodiment, the degree of reduced immunostimulatory response isselected for optimized RNAi activity. For example, retaining a certaindegree of immunostimulation can be preferred to treat viral infection,where less than 100% reduction in immunostimulation may be preferred formaximal antiviral activity (e.g., about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% reduction in immunostimulation) whereas the inhibitionof expression of an endogenous gene target may be preferred with siNAmolecules that posses minimal immunostimulatory properties to preventnon-specific toxicity or off target effects (e.g., about 90% to about100% reduction in immunostimulation).

In one embodiment, a formulated siNA composition of the invention isdesigned such that the composition is not toxic to cells or has aminimized toxicicological profile such that the composition does notinterfere with the efficacy of RNAi mediated by the siNA component ofthe formulated siNA composition or result in toxicity to the cells.

The term “biologically active molecule” as used herein refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive molecules include antibodies (e.g., monoclonal, chimeric,humanized etc.), cholesterol, hormones, antivirals, peptides, proteins,chemotherapeutics, antibiotics, small molecules, vitamins, co-factors,nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids,antisense nucleic acids, triplex forming oligonucleotides, 2,5-Achimeras, siNA, siRNA, miRNA, RNAi inhibitors, dsRNA, allozymes,aptamers, decoys and analogs thereof. Biologically active molecules ofthe invention also include molecules capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules, for example, lipids and polymers such as polyamines,polyamides, polyethylene glycol and other polyethers. In certainembodiments, the term biologically active molecule is usedinterchangeably with the term “molecule” or “molecule of interest”herein.

The term “carrier” or “carrier molecule” as used herein refers to anycompound or composition that can potentiate the activity and/orintracellular delivery of a biologically active molecule by associationwith a delivery vehicle or system. Not wishing to be bound by anymechanism or theory, the carrier molecule provides for maximizedefficiency of a delivery vehicle or system which enables potentintracellular delivery of a biologically active molecule, allowing for areduced amount or concentration of the biologically active material toimpart biologic activity in a cell, tissue, or organism compared to useof the delivery vehicle or system without the carrier molecule.Alternately, the carrier molecule provides for maximized activity of thebiologically active molecule through interation with one or more factorsthat impart biologic activity of the biologically active molecule andthereby potentiate the activity of the biologically active molecule. Inone embodiment, the carrier molecule is used to displace or replace aspecified amount of the biologically active molecule in association withthe delivery vehicle or system. In one embodiment, between about 1 andabout 99 percent (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent) of themolar mass, molecular weight, or concentration of the biologicallyactive molecule can be replaced or displaced with the carrier moleculein or in association with the delivery vehicle or system. Non-limitingexamples of carrier molecules include lipids (e.g., cationic lipids,neutral lipids), peptides, proteins, steroids (e.g., cholesterol,estrogen, testosterone, progesterone, glucocortisone, adrenaline,insulin, glucagon, cortisol, vitamin D, thyroid hormone, retinoic acid,and/or growth hormones), small molecules, vitamins, co-factors,nucleosides, nucleotides, polynucleotides (e.g., single, double, ortriple stranded), polymers, albumin, collagen, and gelatin,polysaccharides such as dextrans and starches, and matrix formingcompositions including polylactide (PLA), polyglycolide (PGA),lactide-glycolide copolymers (PLG), poly(lactic-co-glycolic acid)(PLGA), polycaprolactone, lactide-caprolactone copolymers,polyhydroxybutyrate, polyalkylcyanoacrylates, polyanhydrides,polyorthoesters, acrylate polymers and copolymers such as methylmethacrylate, methacrylic acid, hydroxyalkyl acrylates andmethacrylates, ethylene glycol dimethacrylate, acrylamide and/orbisacrylamide, cellulose-based polymers, ethylene glycol polymers andcopolymers, oxyethylene and oxypropylene polymers, poly(vinyl alcohol),polyvinylacetate, polyvinylpyrrolidone, polyvinylpyridine, and/or anycombination thereof. In one embodiment, a polynucleotide based carriermolecule of the invention comprises one or more nucleic acid molecules,including single stranded RNA or DNA molecules, for example from about 2to about 100,000 bases in length; double stranded RNA or DNA molecules,for example from about 2 to about 100,000 base pairs in length, ortriplex RNA or DNA molecules, for example from about 2 to about 100,000base pairs in length. In one embodiment, a polynucleotide based carriermolecule of the invention comprises a non-human DNA derived from adivergent species, such as non-human sperm DNA (see for exampleJP63102682, describing salmon sperm DNA). In another embodiment, apolynucleotide based carrier molecule of the invention comprises anon-human RNA derived from a divergent species, such as non-human tRNA.In one embodiment, a polynucleotide carrier molecule is a shortinterfering nucleic acid (siNA) molecule as described herein. In anotherembodiment, a polynucleotide carrier molecule is not complementary to atarget nucleic acid molecule which is targeted by a biologically activemolecule within the same composition. For example, if a biologicallyactive molecule of the invention comprises a siNA molecule that hascomplementarity to a target polynucleotide sequence, then a nucleic acidbased carrier molecule utilized in a composition of the invention wouldcomprise sequence that does not have complementarity to the targetpolynucleotide sequence. In one embodiment, the carrier molecule of theinvention is a component of a formulation of the invention. In oneembodiment, the carrier molecule of the invention is devoid ofpolynucleotide.

The term “vehicle” as used herein refers to any delivery system orcomposition that is capable of transporting a biologically activemolecule. Non-limiting examples of vehicles include transfection agents,liposomes, microparticles, nanoparticles, capsids, viroids, virions,virus like particles (VLP), protein cages, ferritins, hydrogels andpolymers; lipid nanoparticle or LNP compositions (see for example TableIV and U.S. Patent Application Publication No. 20060240554 and U.S. Ser.No. 11/586,102, filed Oct. 24, 2006); stable nucleic acid particle orSNALP compositions (see for example International PCT Publication No.WO2007012191, and U.S. Patent Application Publication Nos. 2006083780,2006051405, US2005175682, US2004142025, US2003077829, US2006240093);delivery systems as described in International PCT Publication Nos.WO2005105152 and WO2007014391, and U.S. Pat. Nos. 7,148,205, 7,144,869,7,138,382, 7,101,995, 7,098,032, 7,098,030, 7,094,605, 7,091,041,7,087,770, 7,071,163, 7,049,144, 7,049,142, 7,045,356, 7,033,607,7,022,525, 7,019,113, 7,015,040, 6,936,729, 6,919,091, 6,897,068,6,881,576, 6,872,519, 6,867,196, 6,818,626, 6,794,189, 6,740,643,6,740,336, 6,706,922, 6,673,612, 6,630,351, 6,627,616, 6,593,465,6,458,382, 6,429,200, 6,383,811, 6,379,966, 6,339,067, 6,265,387,6,262,252, 6,180,784, 6,126,964, 6,093,701, and 5,744,335; peptides orpeptide related delivery systems (see for example U.S. PatentApplication Publication Nos. 20060040882, 20050136437, 20050031549, and20060062758); proteins such as albumin, collagen, and gelatin;polysaccharides such as dextrans and starches, and matrix formingcompositions including polylactide (PLA), polyglycolide (PGA),lactide-glycolide copolymers (PLG), poly(lactic-co-glycolic acid)(PLGA), polycaprolactone, lactide-caprolactone copolymers,polyhydroxybutyrate, polyalkylcyanoacrylates, polyanhydrides,polyorthoesters, acrylate polymers and copolymers such as methylmethacrylate, methacrylic acid, hydroxyalkyl acrylates andmethacrylates, ethylene glycol dimethacrylate, acrylamide and/orbisacrylamide, cellulose-based polymers, ethylene glycol polymers andcopolymers, oxyethylene and oxypropylene polymers, poly(vinyl alcohol),polyvinylacetate, polyvinylpyrrolidone, polyvinylpyridine, and/or anycombination thereof. In certain embodiments herein, the term vehicle isused interchangeably with the term formulation.

The term “lipid nanoparticle”, or “lipid nanoparticle composition” or“LNP” as used herein refers to a composition comprising one or morecarrier molecules and/or one or more biologically active moleculesindependently or in combination with a cationic lipid, a neutral lipid,and/or a polyethyleneglycol-diacylglycerol (i.e., polyethyleneglycoldiacylglycerol (PEG-DAG), PEG-cholesterol, or PEG-DMB) conjugate. Aformulation or composition can further comprise cholesterol or acholesterol derivative (see FIG. 12). The cationic lipid of theinvention can comprise a compound having any of Formulae CLI, CLII, CLM,CLIV, CLV, CLVI, CLVII, CLVIII, CLIX, CLX, CLXI, CLXII, CLXIII, CLXIV,CLXV, CLXVI, CLXVII, CLXVIII, CLXIX, CLXX, CLXXI, CLXXII, CLXXIII,CLXXIV, CLXXV, CLXXVI, CLXXVII, CLXXVIII, CLXXIX, CLXXX, CLXXXI,CLXXXII, CLXXXIII, CLXXXIV, CLXXXV, CLXXXVI, CLXXXVII, CLXXXVIII,CLXXXIX, CLXXXX, CLXXXXI, CLXXXXII, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-Dioleoyl-3-Dimethylammonium-propane (DODAP),1,2-Dioleoylcarbamyl-3-Dimethylammonium-propane (DOCDAP),1,2-Dilineoyl-3-Dimethylammonium-propane (DLINDAP),3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA),2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,12′-octadecadienoxy)propane (CpLin DMA),N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA) and/or a mixture thereof.The neutral lipid can comprise dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine(EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and/or amixture thereof. The PEG conjugate can comprise a PEG-dilaurylglycerol(C12), a PEG-dimyristylglycerol (C14), a PEG-dipalmitoylglycerol (C16),a PEG-disterylglycerol (C18), PEG-dilaurylglycamide (C12),PEG-dimyristylglycamide (C14), PEG-dipalmitoylglycamide (C16),PEG-disterylglycamide (C18), PEG-cholesterol, or PEG-DMB. The cationiclipid component can comprise from about 2% to about 60%, from about 5%to about 45%, from about 5% to about 15%, or from about 40% to about 50%of the total lipid present in the formulation. The neutral lipidcomponent can comprise from about 5% to about 90%, or from about 20% toabout 85% of the total lipid present in the formulation. The PEG-DAGconjugate (e.g., polyethyleneglycol diacylglycerol (PEG-DAG),PEG-cholesterol, or PEG-DMB) can comprise from about 1% to about 20%, orfrom about 4% to about 15% of the total lipid present in theformulation. The cholesterol component can comprise from about 10% toabout 60%, or from about 20% to about 45% of the total lipid present inthe formulation. In one embodiment, a formulation or composition of theinvention comprises a cationic lipid component comprising about 7.5% ofthe total lipid present in the formulation, a neutral lipid comprisingabout 82.5% of the total lipid present in the formulation, and a PEGconjugate comprising about 10% of the total lipid present in theformulation. In one embodiment, a formulation or composition of theinvention comprises a biologically active molecule, DODMA, DSPC, and aPEG-DAG conjugate. In one embodiment, the PEG-DAG conjugate isPEG-dilaurylglycerol (C12), PEG-dimyristylglycerol (C14),PEG-dipalmitoylglycerol (C16), or PEG-disterylglycerol (C18). In anotherembodiment, the formulation or composition also comprises cholesterol ora cholesterol derivative. In one embodiment, the formulation orcomposition comprises a lipid nanoparticle formulation as shown in TableIV. In one embodiment, the LNP comprises a formulated siNA composition.In another embodiment, the LNP comprises a formulated miRNA composition.In another embodiment, the LNP comprises a formulated RNAi inhibitorcomposition.

The term “formulated siNA composition” as used herein refers to acomposition comprising one or more siNA molecules or a vector encodingone or more siNA molecules independently or in combination with acationic lipid, a neutral lipid, and/or apolyethyleneglycol-diacylglycerol (PEG-DAG) or PEG-cholesterol(PEG-Chol) conjugate. A formulated siNA composition can further comprisecholesterol or a cholesterol derivative. The cationic lipid of theinvention can comprise a compound having any of Formulae CLI, CLII,CLIII, CLIV, CLV, CLVI, CLVII, CLVIII, CLIX, CLX, CLXI, CLXII, CLXIII,CLXIV, CLXV, CLXVI, CLXVII, CLXVIII, CLXIX, CLXX, CLXXI, CLXXII,CLXXIII, CLXXIV, CLXXV, CLXXVI, CLXXVII, CLXXVIII, CLXXIX, CLXXX,CLXXXI, CLXXXII, CLXXXIII, CLXXXIV, CLXXXV, CLXXXVI, CLXXXVII,CLXXXVIII, CLXXXIX, CLXXXX, CLXXXXI, CLXXXXII,N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-Dioleoyl-3-Dimethylammonium-propane (DODAP),1,2-Dioleoylcarbamyl-3-Dimethylammonium-propane (DOCDAP),1,2-Dilineoyl-3-Dimethylammonium-propane (DLINDAP),3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA),2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,12′-octadecadienoxy)propane(CpLin DMA), N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA) and/or amixture thereof. The neutral lipid can comprise a compound having any ofFormulae NLI-NLVII, dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine(EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and/or amixture thereof. The PEG conjugate can comprise a PEG-dilaurylglycerol(C12), a PEG-dimyristylglycerol (C14), a PEG-dipalmitoylglycerol (C16),a PEG-disterylglycerol (C18), PEG-dilaurylglycamide (C12),PEG-dimyristylglycamide (C14), PEG-dipalmitoylglycamide (C16),PEG-disterylglycamide (C18), PEG-cholesterol, or PEG-DMB. The cationiclipid component can comprise from about 2% to about 60%, from about 5%to about 45%, from about 5% to about 15%, or from about 40% to about 50%of the total lipid present in the formulation. The neutral lipidcomponent can comprise from about 5% to about 90%, or from about 20% toabout 85% of the total lipid present in the formulation. The PEG-DAGconjugate can comprise from about 1% to about 20%, or from about 4% toabout 15% of the total lipid present in the formulation. The cholesterolcomponent can comprise from about 10% to about 60%, or from about 20% toabout 45% of the total lipid present in the formulation. In oneembodiment, a formulated siNA composition of the invention comprises acationic lipid component comprising about 7.5% of the total lipidpresent in the formulation, a neutral lipid comprising about 82.5% ofthe total lipid present in the formulation, and a PEG-DAG conjugatecomprising about 10% of the total lipid present in the formulation. Inone embodiment, a formulated siNA composition of the invention comprisesa siNA molecule, DODMA, DSPC, and a PEG-DAG conjugate. In oneembodiment, the PEG-DAG conjugate is PEG-dilaurylglycerol (C12),PEG-dimyristylglycerol (C14), PEG-dipalmitoylglycerol (C16), orPEG-disterylglycerol (C18). In another embodiment, the formulated siNAcomposition also comprises cholesterol or a cholesterol derivative.

The term “formulated miRNA composition” as used herein refers to acomposition comprising one or more miRNA molecules or a vector encodingone or more miRNA molecules independently or in combination with acationic lipid, a neutral lipid, and/or apolyethyleneglycol-diacylglycerol (PEG-DAG) or PEG-cholesterol(PEG-Chol) conjugate. A formulated miRNA composition can furthercomprise cholesterol or a cholesterol derivative. The cationic lipid ofthe invention can comprise a compound having any of Formulae CLI, CLII,CLIII, CLIV, CLV, CLVI, CLVII, CLVIII, CLIX, CLX, CLXI, CLXII, CLXIII,CLXIV, CLXV, CLXVI, CLXVII, CLXVIII, CLXIX, CLXX, CLXXI, CLXXII,CLXXIII, CLXXIV, CLXXV, CLXXVI, CLXXVII, CLXXVIII, CLXXIX, CLXXX,CLXXXI, CLXXXII, CLXXXIII, CLXXXIV, CLXXXV, CLXXXVI, CLXXXVII,CLXXXVIII, CLXXXIX, CLXXXX, CLXXXXI, CLXXXXII,N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-Dioleoyl-3-Dimethylammonium-propane (DODAP),1,2-Dioleoylcarbamyl-3-Dimethylammonium-propane (DOCDAP),1,2-Dilineoyl-3-Dimethylammonium-propane (DLINDAP),3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA),2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′, 12′-octadecadienoxy)propane (CpLin DMA),N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA) and/or a mixture thereof.The neutral lipid can comprise a compound having any of FormulaeNLI-NLVII, dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine(EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and/or amixture thereof. The PEG conjugate can comprise a PEG-dilaurylglycerol(C12), a PEG-dimyristylglycerol (C14), a PEG-dipalmitoylglycerol (C16),a PEG-disterylglycerol (C18), PEG-dilaurylglycamide (C12),PEG-dimyristylglycamide (C14), PEG-dipalmitoylglycamide (C16),PEG-disterylglycamide (C18), PEG-cholesterol, or PEG-DMB. The cationiclipid component can comprise from about 2% to about 60%, from about 5%to about 45%, from about 5% to about 15%, or from about 40% to about 50%of the total lipid present in the formulation. The neutral lipidcomponent can comprise from about 5% to about 90%, or from about 20% toabout 85% of the total lipid present in the formulation. The PEG-DAGconjugate can comprise from about 1% to about 20%, or from about 4% toabout 15% of the total lipid present in the formulation. The cholesterolcomponent can comprise from about 10% to about 60%, or from about 20% toabout 45% of the total lipid present in the formulation. In oneembodiment, a formulated miRNA composition of the invention comprises acationic lipid component comprising about 7.5% of the total lipidpresent in the formulation, a neutral lipid comprising about 82.5% ofthe total lipid present in the formulation, and a PEG-DAG conjugatecomprising about 10% of the total lipid present in the formulation. Inone embodiment, a formulated miRNA composition of the inventioncomprises a miRNA molecule, DODMA, DSPC, and a PEG-DAG conjugate. In oneembodiment, the PEG-DAG conjugate is PEG-dilaurylglycerol (C12),PEG-dimyristylglycerol (C14), PEG-dipalmitoylglycerol (C16), orPEG-disterylglycerol (C18). In another embodiment, the formulated miRNAcomposition also comprises cholesterol or a cholesterol derivative.

The term “formulated RNAi inhibitor composition” as used herein refersto a composition comprising one or more RNAi inhibitor molecules or avector encoding one or more RNAi inhibitor molecules independently or incombination with a cationic lipid, a neutral lipid, and/or apolyethyleneglycol-diacylglycerol (PEG-DAG) or PEG-cholesterol(PEG-Chol) conjugate. A formulated RNAi inhibitor composition canfurther comprise cholesterol or a cholesterol derivative. The cationiclipid of the invention can comprise a compound having any of FormulaeCLI, CLII, CLIII, CLIV, CLV, CLVI, CLVII, CLVIII, CLIX, CLX, CLXI,CLXII, CLXIII, CLXIV, CLXV, CLXVI, CLXVII, CLXVI, CLXIX, CLXX, CLXXI,CLXXII, CLXXIII, CLXXIV, CLXXV, CLXXVI, CLXXVII, CLXXVIII, CLXXIX,CLXXX, CLXXXI, CLXXXII, CLXXXIII, CLXXXIV, CLXXXV, CLXXXVI, CLXXXVII,CLXXXVIII, CLXXXIX, CLXXXX, CLXXXXI, CLXXXXII,N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-Dioleoyl-3-Dimethylammonium-propane (DODAP),1,2-Dioleoylcarbamyl-3-Dimethylammonium-propane (DOCDAP),1,2-Dilineoyl-3-Dimethylammonium-propane (DLINDAP),3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA),2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,12′-octadecadienoxy)propane (CpLin DMA),N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA) and/or a mixture thereof.The neutral lipid can comprise a compound having any of FormulaeNLI-NLVII, dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine(EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and/or amixture thereof. The PEG conjugate can comprise a PEG-dilaurylglycerol(C12), a PEG-dimyristylglycerol (C14), a PEG-dipalmitoylglycerol (C16),a PEG-disterylglycerol (C18), PEG-dilaurylglycamide (C12),PEG-dimyristylglycamide (C14), PEG-dipalmitoylglycamide (C16),PEG-disterylglycamide (C18), PEG-cholesterol, or PEG-DMB. The cationiclipid component can comprise from about 2% to about 60%, from about 5%to about 45%, from about 5% to about 15%, or from about 40% to about 50%of the total lipid present in the formulation. The neutral lipidcomponent can comprise from about 5% to about 90%, or from about 20% toabout 85% of the total lipid present in the formulation. The PEG-DAGconjugate can comprise from about 1% to about 20%, or from about 4% toabout 15% of the total lipid present in the formulation. The cholesterolcomponent can comprise from about 10% to about 60%, or from about 20% toabout 45% of the total lipid present in the formulation. In oneembodiment, a formulated RNAi inhibitor composition of the inventioncomprises a cationic lipid component comprising about 7.5% of the totallipid present in the formulation, a neutral lipid comprising about 82.5%of the total lipid present in the formulation, and a PEG-DAG conjugatecomprising about 10% of the total lipid present in the formulation. Inone embodiment, a formulated RNAi inhibitor composition of the inventioncomprises a RNAi inhibitor molecule, DODMA, DSPC, and a PEG-DAGconjugate. In one embodiment, the PEG-DAG conjugate isPEG-dilaurylglycerol (C12), PEG-dimyristylglycerol (C14),PEG-dipalmitoylglycerol (C16), or PEG-disterylglycerol (C18). In anotherembodiment, the formulated RNAi inhibitor composition also comprisescholesterol or a cholesterol derivative.

By “cationic lipid” as used herein is meant any lipophilic compoundhaving cationic change at a specified pH, such as a compound having anyof Formulae CLI-CLXXXXVI.

By “neutral lipid” as used herein is meant any lipophilic compoundhaving non-cationic change (e.g., anionic or neutral charge) at aspecified pH.

By “PEG” is meant, any polyethylene glycol or other polyalkylene etheror equivalent polymer. In one embodiment, the PEG is a PEG conjugatewhich can comprise a 200 to 10,000 atom PEG molecule linked to, orexample, a lipid moiety of the invention. In one embodiment, the PEG isa polydispersion represented by the formula PEG_(n), where n=about 33 to67 for a 1500 Da to 3000 Da PEG, average=45 for 2 KPEG/PEG2000.

By “nanoparticle” is meant a microscopic particle whose size is measuredin nanometers. Nanoparticles of the invention typically range from about1 to about 999 nm in diameter, and can include an encapsulated orenclosed biologically active molecule.

By “microparticle” is meant a is a microscopic particle whose size ismeasured in micrometers. Microparticles of the invention typically rangefrom about 1 to about 100 micrometers in diameter, and can include anencapsulated or enclosed biologically active molecule.

The terms “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, and “chemically-modified shortinterfering nucleic acid molecule” as used herein refer to any nucleicacid molecule capable of inhibiting or down regulating gene expressionor viral replication by mediating RNA interference “RNAi” or genesilencing in a sequence-specific manner (see PCT/US 2004/106390 (WO05/19453), U.S. Ser. No. 10/444,853, filed May 23, 2003 U.S. Ser. No.10/923,536 filed Aug. 20, 2004, U.S. Ser. No. 11/234,730, filed Sep. 23,2005, U.S. Ser. No. 11/299,254, filed Dec. 8, 2005, or PCT/US06/32168,filed Aug. 17, 2006, all incorporated by reference in their entiretiesherein). These terms can refer to both individual nucleic acidmolecules, a plurality of such nucleic acid molecules, or pools of suchnucleic acid molecules. The siNA can be a double-stranded nucleic acidmolecule comprising self-complementary sense and antisense regions,wherein the antisense region comprises nucleotide sequence that iscomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof and the sense region having nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof.The siNA can be assembled from two separate oligonucleotides, where onestrand is the sense strand and the other is the antisense strand,wherein the antisense and sense strands are self-complementary (i.e.,each strand comprises nucleotide sequence that is complementary tonucleotide sequence in the other strand; such as where the antisensestrand and sense strand form a duplex or double stranded structure, forexample wherein the double stranded region is about 15 to about 30,e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29or 30 base pairs; the antisense strand comprises nucleotide sequencethat is complementary to nucleotide sequence in a target nucleic acidmolecule or a portion thereof and the sense strand comprises nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof (e.g., about 15 to about 25 or more nucleotides of the siNAmolecule are complementary to the target nucleic acid or a portionthereof). Alternatively, the siNA is assembled from a singleoligonucleotide, where the self-complementary sense and antisenseregions of the siNA are linked by means of a nucleic acid based ornon-nucleic acid-based linker(s). The siNA can be a polynucleotide witha duplex, asymmetric duplex, hairpin or asymmetric hairpin secondarystructure, having self-complementary sense and antisense regions,wherein the antisense region comprises nucleotide sequence that iscomplementary to nucleotide sequence in a separate target nucleic acidmolecule or a portion thereof and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. The siNA can be a circular single-stranded polynucleotidehaving two or more loop structures and a stem comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises nucleotide sequence that is complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof and thesense region having nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof, and wherein the circularpolynucleotide can be processed either in vivo or in vitro to generatean active siNA molecule capable of mediating RNAi. The siNA can alsocomprise a single stranded polynucleotide having nucleotide sequencecomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof (for example, where such siNA molecule does notrequire the presence within the siNA molecule of nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof),wherein the single stranded polynucleotide can further comprise aterminal phosphate group, such as a 5′-phosphate (see for exampleMartinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002,Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certainembodiments, the siNA molecule of the invention comprises separate senseand antisense sequences or regions, wherein the sense and antisenseregions are covalently linked by nucleotide or non-nucleotide linkersmolecules as is known in the art, or are alternately non-covalentlylinked by ionic interactions, hydrogen bonding, van der waalsinteractions, hydrophobic interactions, and/or stacking interactions. Incertain embodiments, the siNA molecules of the invention comprisenucleotide sequence that is complementary to nucleotide sequence of atarget gene. In another embodiment, the siNA molecule of the inventioninteracts with nucleotide sequence of a target gene in a manner thatcauses inhibition of expression of the target gene. As used herein, siNAmolecules need not be limited to those molecules containing only RNA,but further encompasses chemically-modified nucleotides andnon-nucleotides. In certain embodiments, the short interfering nucleicacid molecules of the invention lack 2′-hydroxy(2′-OH) containingnucleotides. Applicant describes in certain embodiments shortinterfering nucleic acids that do not require the presence ofnucleotides having a 2′-hydroxy group for mediating RNAi and as such,short interfering nucleic acid molecules of the invention optionally donot include any ribonucleotides (e.g., nucleotides having a 2′-OHgroup). Such siNA molecules that do not require the presence ofribonucleotides within the siNA molecule to support RNAi can howeverhave an attached linker or linkers or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides atabout 5, 10, 20, 30, 40, or 50% of the nucleotide positions. Themodified short interfering nucleic acid molecules of the invention canalso be referred to as short interfering modified oligonucleotides“siMON.” As used herein, the term siNA is meant to be equivalent toother terms used to describe nucleic acid molecules that are capable ofmediating sequence specific RNAi, for example short interfering RNA(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpinRNA (shRNA), short interfering oligonucleotide, short interferingnucleic acid, short interfering modified oligonucleotide,chemically-modified siRNA, post-transcriptional gene silencing RNA(ptgsRNA), and others. Non limiting examples of siNA molecules of theinvention are shown in U.S. Ser. No. 11/234,730, filed Sep. 23, 2005,incorporated by reference in its entirety herein. Such siNA moleculesare distinct from other nucleic acid technologies known in the art thatmediate inhibition of gene expression, such as ribozymes, antisense,triplex forming, aptamer, 2,5-A chimera, or decoy oligonucleotides.

By “RNA interference” or “RNAi” is meant a biological process ofinhibiting or down regulating gene expression in a cell as is generallyknown in the art and which is mediated by short interfering nucleic acidmolecules, see for example Zamore and Haley, 2005, Science, 309,1519-1524; Vaughn and Martienssen, 2005, Science, 309, 1525-1526; Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429;Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PCTPublication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00/01846; Mello and Fire, International PCTPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99/07409; and Li et al., International PCTPublication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819;Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science,297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002,RNA, 8, 842-850; Reinhart et al., 2002, gene & Dev., 16, 1616-1626; andReinhart & Bartel, 2002, Science, 297, 1831). In addition, as usedherein, the term RNAi is meant to be equivalent to other terms used todescribe sequence specific RNA interference, such as posttranscriptional gene silencing, translational inhibition,transcriptional inhibition, or epigenetics. For example, siNA moleculesof the invention can be used to epigenetically silence genes at both thepost-transcriptional level or the pre-transcriptional level. In anon-limiting example, epigenetic modulation of gene expression by siNAmolecules of the invention can result from siNA mediated modification ofchromatin structure or methylation patterns to alter gene expression(see, for example, Verdel et al., 2004, Science, 303, 672-676;Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science,297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,2232-2237). In another non-limiting example, modulation of geneexpression by siNA molecules of the invention can result from siNAmediated cleavage of RNA (either coding or non-coding RNA) via RISC, oralternately, translational inhibition as is known in the art. In anotherembodiment, modulation of gene expression by siNA molecules of theinvention can result from transcriptional inhibition (see for exampleJanowski et al., 2005, Nature Chemical Biology, 1, 216-222).

By “asymmetric hairpin” as used herein is meant a linear siNA moleculecomprising an antisense region, a loop portion that can comprisenucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complementary nucleotides to base pair with theantisense region and form a duplex with loop. For example, an asymmetrichairpin siNA molecule of the invention can comprise an antisense regionhaving length sufficient to mediate RNAi in a cell or in vitro system(e.g. about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 nucleotides) and a loop region comprisingabout 4 to about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, or 12)nucleotides, and a sense region having about 3 to about 25 (e.g., about3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25) nucleotides that are complementary to the antisenseregion. The asymmetric hairpin siNA molecule can also comprise a5′-terminal phosphate group that can be chemically modified. The loopportion of the asymmetric hairpin siNA molecule can comprisenucleotides, non-nucleotides, linker molecules, or conjugate moleculesas described herein.

By “asymmetric duplex” as used herein is meant a siNA molecule havingtwo separate strands comprising a sense region and an antisense region,wherein the sense region comprises fewer nucleotides than the antisenseregion to the extent that the sense region has enough complementarynucleotides to base pair with the antisense region and form a duplex.For example, an asymmetric duplex siNA molecule of the invention cancomprise an antisense region having length sufficient to mediate RNAi ina cell or in vitro system (e.g. about 15 to about 30, or about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides)and a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25) nucleotides that are complementary to the antisense region.

The term “polynucleotide” or “nucleic acid molecule” as used herein,refers to a molecule having nucleotides. The nucleic acid can be single,double, or multiple stranded and can comprise modified or unmodifiednucleotides or non-nucleotides or various mixtures and combinationsthereof.

By “RNAi inhibitor” is meant any molecule that can down regulate, reduceor inhibit RNA interference function or activity in a cell or organism.An RNAi inhibitor can down regulate, reduce or inhibit RNAi (e.g., RNAimediated cleavage of a target polynucleotide, translational inhibition,or transcriptional silencing) by interaction with or interfering thefunction of any component of the RNAi pathway, including proteincomponents such as RISC, or nucleic acid components such as miRNAs orsiRNAs. A RNAi inhibitor can be a siNA molecule, an antisense molecule,an aptamer, or a small molecule that interacts with or interferes withthe function of RISC, a miRNA, or a siRNA or any other component of theRNAi pathway in a cell or organism. By inhibiting RNAi (e.g., RNAimediated cleavage of a target polynucleotide, translational inhibition,or transcriptional silencing), a RNAi inhibitor of the invention can beused to modulate (e.g, up-regulate or down regulate) the expression of atarget gene. In one embodiment, a RNA inhibitor of the invention is usedto up-regulate gene expression by interfering with (e.g., reducing orpreventing) endogenous down-regulation or inhibition of gene expressionthrough translational inhibition, transcriptional silencing, or RISCmediated cleavage of a polynucleotide (e.g., mRNA). By interfering withmechanisms of endogenous repression, silencing, or inhibition of geneexpression, RNAi inhibitors of the invention can therefore be used toup-regulate gene expression for the treatment of diseases, traits, orconditions resulting from a loss of function. In one embodiment, theterm “RNAi inhibitor” is used in place of the term “siNA” in the variousembodiments herein, for example, with the effect of increasing geneexpression for the treatment of loss of function diseases, traits,and/or conditions.

The term “enzymatic nucleic acid molecule” as used herein refers to anucleic acid molecule which has complementarity in a substrate bindingregion to a specified gene target, and also has an enzymatic activitywhich is active to specifically cleave target RNA. That is, theenzymatic nucleic acid molecule is able to intermolecularly cleave RNAand thereby inactivate a target RNA molecule. These complementaryregions allow sufficient hybridization of the enzymatic nucleic acidmolecule to the target RNA and thus permit cleavage. One hundred percentcomplementarity is preferred, but complementarity as low as 50-75% canalso be useful in this invention (see for example Werner and Uhlenbeck,1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999,Antisense and Nucleic Acid Drug Dev., 9, 25-31). The nucleic acids canbe modified at the base, sugar, and/or phosphate groups. The termenzymatic nucleic acid is used interchangeably with phrases such asribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme oraptamer-binding ribozyme, regulatable ribozyme, catalyticoligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease,endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of theseterminologies describe nucleic acid molecules with enzymatic activity.The specific enzymatic nucleic acid molecules described in the instantapplication are not limiting in the invention and those skilled in theart will recognize that all that is important in an enzymatic nucleicacid molecule of this invention is that it has a specific substratebinding site which is complementary to one or more of the target nucleicacid regions, and that it have nucleotide sequences within orsurrounding that substrate binding site which impart a nucleic acidcleaving and/or ligation activity to the molecule (Cech et al., U.S.Pat. No. 4,987,071; Cech et al., 1988, 260 JAMA 3030). Ribozymes andenzymatic nucleic molecules of the invention can be chemically modifiedas is generally known in the art or as described herein.

The term “antisense nucleic acid”, as used herein, refers to anon-enzymatic nucleic acid molecule that binds to target RNA by means ofRNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993Nature 365, 566) interactions and alters the activity of the target RNA(for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf etal., U.S. Pat. No. 5,849,902). Typically, antisense molecules arecomplementary to a target sequence along a single contiguous sequence ofthe antisense molecule. However, in certain embodiments, an antisensemolecule can bind to substrate such that the substrate molecule forms aloop, and/or an antisense molecule can bind such that the antisensemolecule forms a loop. Thus, the antisense molecule can be complementaryto two (or even more) non-contiguous substrate sequences or two (or evenmore) non-contiguous sequence portions of an antisense molecule can becomplementary to a target sequence or both. For a review of currentantisense strategies, see Schmajuk et al., 1999, J. Biol. Chem., 274,21783-21789, Delihas et al., 1997, Nature, 15, 751-753, Stein et al.,1997, Antisense N. A. Drug Dev., 7, 151, Crooke, 2000, Methods Enzymol.,313, 3-45; Crooke, 1998, Biotech. Genet. Eng. Rev., 15, 121-157, Crooke,1997, Ad. Pharmacol., 40, 1-49. In addition, antisense DNA can be usedto target RNA by means of DNA-RNA interactions, thereby activating RNaseH, which digests the target RNA in the duplex. The antisenseoligonucleotides can comprise one or more RNAse H activating region,which is capable of activating RNAse H cleavage of a target RNA.Antisense DNA can be synthesized chemically or expressed via the use ofa single stranded DNA expression vector or equivalent thereof. Antisensemolecules of the invention can be chemically modified as is generallyknown in the art or as described herein.

The term “RNase H activating region” as used herein, refers to a region(generally greater than or equal to 4-25 nucleotides in length,preferably from 5-11 nucleotides in length) of a nucleic acid moleculecapable of binding to a target RNA to form a non-covalent complex thatis recognized by cellular RNase H enzyme (see for example Arrow et al.,U.S. Pat. No. 5,849,902; Arrow et al., U.S. Pat. No. 5,989,912). TheRNase H enzyme binds to the nucleic acid molecule-target RNA complex andcleaves the target RNA sequence. The RNase H activating regioncomprises, for example, phosphodiester, phosphorothioate (preferably atleast four of the nucleotides are phosphorothiote substitutions; morespecifically, 4-11 of the nucleotides are phosphorothiotesubstitutions); phosphorodithioate, 5′-thiophosphate, ormethylphosphonate backbone chemistry or a combination thereof. Inaddition to one or more backbone chemistries described above, the RNaseH activating region can also comprise a variety of sugar chemistries.For example, the RNase H activating region can comprise deoxyribose,arabino, fluoroarabino or a combination thereof, nucleotide sugarchemistry. Those skilled in the art will recognize that the foregoingare non-limiting examples and that any combination of phosphate, sugarand base chemistry of a nucleic acid that supports the activity of RNaseH enzyme is within the scope of the definition of the RNase H activatingregion and the instant invention.

The term “2-5A antisense chimera” as used herein, refers to an antisenseoligonucleotide containing a 5′-phosphorylated 2′-5′-linked adenylateresidue. These chimeras bind to target RNA in a sequence-specific mannerand activate a cellular 2-5A-dependent ribonuclease which, in turn,cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA90, 1300; Silverman et al., 2000, Methods Enzymol., 313, 522-533; Playerand Torrence, 1998, Pharmacol. Ther., 78, 55-113). 2-5A antisensechimera molecules of the invention can be chemically modified as isgenerally known in the art or as described herein.

The term “triplex forming oligonucleotides” as used herein, refers to anoligonucleotide that can bind to a double-stranded DNA in asequence-specific manner to form a triple-strand helix. Formation ofsuch triple helix structure has been shown to inhibit transcription ofthe targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci.USA 89, 504; Fox, 2000, Curr. Med. Chem., 7, 17-37; Praseuth et. al.,2000, Biochim. Biophys. Acta, 1489, 181-206). Triplex formingoligonucleotide molecules of the invention can be chemically modified asis generally known in the art or as described herein.

The term “decoy RNA” as used herein, refers to a RNA molecule or aptamerthat is designed to preferentially bind to a predetermined ligand. Suchbinding can result in the inhibition or activation of a target molecule.The decoy RNA or aptamer can compete with a naturally occurring bindingtarget for the binding of a specific ligand. For example, it has beenshown that over-expression of HIV trans-activation response (TAR) RNAcan act as a “decoy” and efficiently binds HIV tat protein, therebypreventing it from binding to TAR sequences encoded in the HIV RNA(Sullenger et al., 1990, Cell, 63, 601-608). This is but a specificexample and those in the art will recognize that other embodiments canbe readily generated using techniques generally known in the art, seefor example Gold et al., 1995, Annu. Rev. Biochem., 64, 763; Brody andGold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2,100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000,Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.Similarly, a decoy RNA can be designed to bind to a receptor and blockthe binding of an effector molecule or a decoy RNA can be designed tobind to receptor of interest and prevent interaction with the receptor.Decoy molecules of the invention can be chemically modified as isgenerally known in the art or as described herein.

The term “single stranded RNA” (ssRNA) as used herein refers to anaturally occurring or synthetic ribonucleic acid molecule comprising alinear single strand, for example a ssRNA can be a messenger RNA (mRNA),transfer RNA (tRNA), ribosomal RNA (rRNA) etc. of a gene.

The term “single stranded DNA” (ssDNA) as used herein refers to anaturally occurring or synthetic deoxyribonucleic acid moleculecomprising a linear single strand, for example, a ssDNA can be a senseor antisense gene sequence or EST (Expressed Sequence Tag).

The term “double stranded RNA” or “dsRNA” as used herein refers to adouble stranded RNA molecule capable of RNA interference, includingshort interfering RNA (siNA).

The term “allozyme” as used herein refers to an allosteric enzymaticnucleic acid molecule, see for example see for example George et al.,U.S. Pat. Nos. 5,834,186 and 5,741,679, Shih et al., U.S. Pat. No.5,589,332, Nathan et al., U.S. Pat. No. 5,871,914, Nathan and Ellington,International PCT publication No. WO 00/24931, Breaker et al.,International PCT Publication Nos. WO 00/26226 and 98/27104, andSullenger et al., International PCT publication No. WO 99/29842.

By “aptamer” or “nucleic acid aptamer” as used herein is meant apolynucleotide that binds specifically to a target molecule wherein thenucleic acid molecule has sequence that is distinct from sequencerecognized by the target molecule in its natural setting. Alternately,an aptamer can be a nucleic acid molecule that binds to a targetmolecule where the target molecule does not naturally bind to a nucleicacid. The target molecule can be any molecule of interest. For example,the aptamer can be used to bind to a ligand-binding domain of a protein,thereby preventing interaction of the naturally occurring ligand withthe protein. This is a non-limiting example and those in the art willrecognize that other embodiments can be readily generated usingtechniques generally known in the art, see for example Gold et al.,1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J.Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser,2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287,820; and Jayasena, 1999, Clinical Chemistry, 45, 1628. Aptamer moleculesof the invention can be chemically modified as is generally known in theart or as described herein.

By “modulate” is meant that the expression of the gene, or level of RNAmolecule or equivalent RNA molecules encoding one or more proteins orprotein subunits, or activity of one or more proteins or proteinsubunits is up regulated or down regulated, such that expression, level,or activity is greater than or less than that observed in the absence ofthe modulator. For example, the term “modulate” can mean “inhibit,” butthe use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that theexpression of the gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more proteins or protein subunits, is reduced below thatobserved in the absence of the nucleic acid molecules (e.g., siNA) ofthe invention. In one embodiment, inhibition, down-regulation orreduction with a siNA molecule is below that level observed in thepresence of an inactive or attenuated molecule. In another embodiment,inhibition, down-regulation, or reduction with siNA molecules is belowthat level observed in the presence of, for example, a siNA moleculewith scrambled sequence or with mismatches. In another embodiment,inhibition, down-regulation, or reduction of gene expression with anucleic acid molecule of the instant invention is greater in thepresence of the nucleic acid molecule than in its absence. In oneembodiment, inhibition, down regulation, or reduction of gene expressionis associated with post transcriptional silencing, such as RNAi mediatedcleavage of a target nucleic acid molecule (e.g. RNA) or inhibition oftranslation. In one embodiment, inhibition, down regulation, orreduction of gene expression is associated with pretranscriptionalsilencing.

By “up-regulate”, or “promote”, it is meant that the expression of thegene, or level of RNA molecules or equivalent RNA molecules encoding oneor more proteins or protein subunits, or activity of one or moreproteins or protein subunits, is increased above that observed in theabsence of the nucleic acid molecules (e.g., siNA) of the invention. Inone embodiment, up-regulation or promotion of gene expression with ansiNA molecule is above that level observed in the presence of aninactive or attenuated molecule. In another embodiment, up-regulation orpromotion of gene expression with siNA molecules is above that levelobserved in the presence of, for example, an siNA molecule withscrambled sequence or with mismatches. In another embodiment,up-regulation or promotion of gene expression with a nucleic acidmolecule of the instant invention is greater in the presence of thenucleic acid molecule than in its absence. In one embodiment,up-regulation or promotion of gene expression is associated withinhibition of RNA mediated gene silencing, such as RNAi mediatedcleavage or silencing of a coding or non-coding RNA target that downregulates, inhibits, or silences the expression of the gene of interestto be up-regulated. The down regulation of gene expression can, forexample, be induced by a coding RNA or its encoded protein, such asthrough negative feedback or antagonistic effects. The down regulationof gene expression can, for example, be induced by a non-coding RNAhaving regulatory control over a gene of interest, for example bysilencing expression of the gene via translational inhibition, chromatinstructure, methylation, RISC mediated RNA cleavage, or translationalinhibition. As such, inhibition or down regulation of targets that downregulate, suppress, or silence a gene of interest can be used toup-regulate or promote expression of the gene of interest towardtherapeutic use.

In one embodiment, a RNAi inhibitor of the invention is used to upregulate gene expression by inhibiting RNAi or gene silencing. Forexample, a RNAi inhibitor of the invention can be used to treat loss offunction diseases and conditions by up-regulating gene expression, suchas in instances of haploinsufficiency where one allele of a particulargene harbors a mutation (e.g., a frameshift, missense, or nonsensemutation) resulting in a loss of function of the protein encoded by themutant allele. In such instances, the RNAi inhibitor can be used to upregulate expression of the protein encoded by the wild type orfunctional allele, thus correcting the haploinsufficiency bycompensating for the mutant or null allele. In another embodiment, asiNA molecule of the invention is used to down regulate expression of atoxic gain of function allele while a RNAi inhibitor of the invention isused concomitantly to up regulate expression of the wild type orfunctional allele, such as in the treatment of diseases, traits, orconditions herein or otherwise known in the art (see for example Rhodeset al., 2004, PNAS USA, 101:11147-11152 and Meisler et al. 2005, TheJournal of Clinical Investigation, 115:2010-2017).

By “gene”, or “target gene”, is meant a nucleic acid that encodes RNA,for example, nucleic acid sequences including, but not limited to,structural genes encoding a polypeptide. A gene or target gene can alsoencode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as smalltemporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA),short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomalRNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Suchnon-coding RNAs can serve as target nucleic acid molecules for siNAmediated RNA interference in modulating the activity of fRNA or ncRNAinvolved in functional or regulatory cellular processes. Abberant fRNAor ncRNA activity leading to disease can therefore be modulated by siNAmolecules of the invention. siNA molecules targeting fRNA and ncRNA canalso be used to manipulate or alter the genotype or phenotype of asubject, organism or cell, by intervening in cellular processes such asgenetic imprinting, transcription, translation, or nucleic acidprocessing (e.g., transamination, methylation etc.). The target gene canbe a gene derived from a cell, an endogenous gene, a transgene, orexogenous genes such as genes of a pathogen, for example a virus, whichis present in the cell after infection thereof. The cell containing thetarget gene can be derived from or contained in any organism, forexample a plant, animal, protozoan, virus, bacterium, or fungus.Non-limiting examples of plants include monocots, dicots, orgymnosperms. Non-limiting examples of animals include vertebrates orinvertebrates. Non-limiting examples of fungi include molds or yeasts.For a review, see for example Snyder and Gerstein, 2003, Science, 300,258-260.

By “target” as used herein is meant, any target protein, peptide, orpolypeptide encoded by a target gene. The term “target” also refers tonucleic acid sequences encoding any target protein, peptide, orpolypeptide having target activity, such as encoded by target RNA. Theterm “target” is also meant to include other target encoding sequence,such as other target isoforms, mutant target genes, splice variants oftarget genes, and target gene polymorphisms. By “target nucleic acid” ismeant any nucleic acid sequence whose expression or activity is to bemodulated. The target nucleic acid can be DNA or RNA.

By “non-canonical base pair” is meant any non-Watson Crick base pair,such as mismatches and/or wobble base pairs, including flippedmismatches, single hydrogen bond mismatches, trans-type mismatches,triple base interactions, and quadruple base interactions. Non-limitingexamples of such non-canonical base pairs include, but are not limitedto, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AAN7 amino, CC 2-carbonyl-amino(H1)-N-3-amino(H2), GA sheared, UC4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AUreverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AAN1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl, GA+carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-amino symmetric,CC carbonyl-amino symmetric, CC N3-amino symmetric, UU 2-carbonyl-iminosymmetric, UU 4-carbonyl-imino symmetric, AA amino-N3, AA N1-amino, ACamino 2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AUN1-imino, AU N3-imino, AU N7-imino, CC carbonyl-amino, GA amino-N1, GAamino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GCcarbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GGcarbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GUimino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl, UC4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H—N3, GAcarbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A)N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonylamino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.

By “target” as used herein is meant, any target protein, peptide, orpolypeptide, such as encoded by Genbank Accession Nos. shown in U.S.Ser. No. 10/923,536 and U.S. Ser. No. 10/923,536, both incorporated byreference herein. The term “target” also refers to nucleic acidsequences or target polynucleotide sequence encoding any target protein,peptide, or polypeptide, such as proteins, peptides, or polypeptidesencoded by sequences having Genbank Accession Nos. shown in U.S. Ser.No. 10/923,536 and U.S. Ser. No. 10/923,536. The target of interest caninclude target polynucleotide sequences, such as target DNA or targetRNA. The term “target” is also meant to include other sequences, such asdiffering isoforms, mutant target genes, splice variants of targetpolynucleotides, target polymorphisms, and non-coding (e.g., ncRNA,miRNA, sRNA) or other regulatory polynucleotide sequences as describedherein. Therefore, in various embodiments of the invention, a doublestranded nucleic acid molecule of the invention (e.g., siNA) havingcomplementarity to a target RNA can be used to inhibit or down regulatemiRNA or other ncRNA activity. In one embodiment, inhibition of miRNA orncRNA activity can be used to down regulate or inhibit gene expression(e.g., gene targets described herein or otherwise known in the art) orviral replication (e.g., viral targets described herein or otherwiseknown in the art) that is dependent on miRNA or ncRNA activity. Inanother embodiment, inhibition of miRNA or ncRNA activity by doublestranded nucleic acid molecules of the invention (e.g. siNA) havingcomplementarity to the miRNA or ncRNA can be used to up regulate orpromote target gene expression (e.g., gene targets described herein orotherwise known in the art) where the expression of such genes is downregulated, suppressed, or silenced by the miRNA or ncRNA. Suchup-regulation of gene expression can be used to treat diseases andconditions associated with a loss of function or haploinsufficiency asare generally known in the art (e.g., muscular dystrophies, cysticfibrosis, or neurologic diseases and conditions described herein such asepilepsy, including severe myoclonic epilepsy of infancy or Dravetsyndrome).

By “homologous sequence” is meant, a nucleotide sequence that is sharedby one or more polynucleotide sequences, such as genes, gene transcriptsand/or non-coding polynucleotides. For example, a homologous sequencecan be a nucleotide sequence that is shared by two or more genesencoding related but different proteins, such as different members of agene family, different protein epitopes, different protein isoforms orcompletely divergent genes, such as a cytokine and its correspondingreceptors. A homologous sequence can be a nucleotide sequence that isshared by two or more non-coding polynucleotides, such as noncoding DNAor RNA, regulatory sequences, introns, and sites of transcriptionalcontrol or regulation. Homologous sequences can also include conservedsequence regions shared by more than one polynucleotide sequence.Homology does not need to be perfect homology (e.g., 100%), as partiallyhomologous sequences are also contemplated by the instant invention(e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%,86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).

By “conserved sequence region” is meant, a nucleotide sequence of one ormore regions in a polynucleotide does not vary significantly betweengenerations or from one biological system, subject, or organism toanother biological system, subject, or organism. The polynucleotide caninclude both coding and non-coding DNA and RNA.

By “sense region” is meant a nucleotide sequence of a siNA moleculehaving complementarity to an antisense region of the siNA molecule. Inaddition, the sense region of a siNA molecule can comprise a nucleicacid sequence having homology with a target nucleic acid sequence. Inone embodiment, the sense region of the siNA molecule is referred to asthe sense strand or passenger strand.

By “antisense region” is meant a nucleotide sequence of a siNA moleculehaving complementarity to a target nucleic acid sequence. In addition,the antisense region of a siNA molecule can optionally comprise anucleic acid sequence having complementarity to a sense region of thesiNA molecule. In one embodiment, the antisense region of the siNAmolecule is referred to as the antisense strand or guide strand.

By “target nucleic acid” or “target polynucleotide” is meant any nucleicacid sequence whose expression or activity is to be modulated. Thetarget nucleic acid can be DNA or RNA. In one embodiment, a targetnucleic acid of the invention is target RNA or DNA.

By “complementary” and “complementarity” (and variations thereof) ismeant to describe a nucleic acid that can form hydrogen bond(s) withanother nucleic acid sequence by either traditional Watson-Crick orother non-traditional types as described herein. Preferably, the degreeof complementarity is such that nucleic acids that are complementaryform double stranded complexes or duplexes under physiologicalconditions. Such nucleic acids can be, but are not necessarily,perfectly complementary. Complementary nucleic acids can include 1, 2,3, or more mismatches so long as the nucleic acids are capable offorming duplexes under physiological conditions. In one embodiment, adouble stranded nucleic acid molecule of the invention, such as an siNAmolecule, wherein each strand is between 15 and 30 nucleotides inlength, comprises between about 10% and about 100% (e.g., about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) complementarity betweenthe two strands of the double stranded nucleic acid molecule. In anotherembodiment, a double stranded nucleic acid molecule of the invention,such as an siNA molecule, where one strand is the sense strand and theother stand is the antisense strand, wherein each strand is between 15and 30 nucleotides in length, comprises between at least about 10% andabout 100% (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 100%) complementarity between the nucleotide sequence in theantisense strand of the double stranded nucleic acid molecule and thenucleotide sequence of its corresponding target nucleic acid molecule,such as a target RNA or target mRNA or viral RNA. In one embodiment, adouble stranded nucleic acid molecule of the invention, such as an siNAmolecule, where one strand comprises nucleotide sequence that isreferred to as the sense region and the other strand comprises anucleotide sequence that is referred to as the antisense region, whereineach strand is between 15 and 30 nucleotides in length, comprisesbetween about 10% and about 100% (e.g., about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 100%) complementarity between the sense regionand the antisense region of the double stranded nucleic acid molecule.In reference to the nucleic molecules of the present invention, thebinding free energy for a nucleic acid molecule with its complementarysequence is sufficient to allow the relevant function of the nucleicacid to proceed, e.g., RNAi activity. Determination of binding freeenergies for nucleic acid molecules is well known in the art (see, e.g.,Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier etal., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987,J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicatesthe percentage of contiguous residues in a nucleic acid molecule thatcan form hydrogen bonds (e.g., Watson-Crick base pairing) with a secondnucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of atotal of 10 nucleotides in the first oligonucleotide being based pairedto a second nucleic acid sequence having 10 nucleotides represents 50%,60%, 70%, 80%, 90%, and 100% complementary respectively). In oneembodiment, a siNA molecule of the invention has perfect complementaritybetween the sense strand or sense region and the antisense strand orantisense region of the siNA molecule. In one embodiment, a siNAmolecule of the invention is perfectly complementary to a correspondingtarget nucleic acid molecule. “Perfectly complementary” means that allthe contiguous residues of a nucleic acid sequence will hydrogen bondwith the same number of contiguous residues in a second nucleic acidsequence. In one embodiment, a siNA molecule of the invention comprisesabout 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides that arecomplementary to one or more target nucleic acid molecules or a portionthereof. In one embodiment, a siNA molecule of the invention has partialcomplementarity (i.e., less than 100% complementarity) between the sensestrand or sense region and the antisense strand or antisense region ofthe siNA molecule or between the antisense strand or antisense region ofthe siNA molecule and a corresponding target nucleic acid molecule. Forexample, partial complementarity can include various mismatches ornon-based paired nucleotides (e.g., 1, 2, 3, 4, 5 or more mismatches ornon-based paired nucleotides) within the siNA structure which can resultin bulges, loops, or overhangs that result between the between the sensestrand or sense region and the antisense strand or antisense region ofthe siNA molecule or between the antisense strand or antisense region ofthe siNA molecule and a corresponding target nucleic acid molecule.

In one embodiment, a double stranded nucleic acid molecule of theinvention, such as siNA molecule, has perfect complementarity betweenthe sense strand or sense region and the antisense strand or antisenseregion of the nucleic acid molecule. In one embodiment, double strandednucleic acid molecule of the invention, such as siNA molecule, isperfectly complementary to a corresponding target nucleic acid molecule.

In one embodiment, double stranded nucleic acid molecule of theinvention, such as siNA molecule, has partial complementarity (i.e.,less than 100% complementarity) between the sense strand or sense regionand the antisense strand or antisense region of the double strandednucleic acid molecule or between the antisense strand or antisenseregion of the nucleic acid molecule and a corresponding target nucleicacid molecule. For example, partial complementarity can include variousmismatches or non-base paired nucleotides (e.g., 1, 2, 3, 4, 5 or moremismatches or non-based paired nucleotides, such as nucleotide bulges)within the double stranded nucleic acid molecule, structure which canresult in bulges, loops, or overhangs that result between the sensestrand or sense region and the antisense strand or antisense region ofthe double stranded nucleic acid molecule or between the antisensestrand or antisense region of the double stranded nucleic acid moleculeand a corresponding target nucleic acid molecule.

In one embodiment, double stranded nucleic acid molecule of theinvention is a microRNA (miRNA). By “mircoRNA” or “miRNA” is meant, asmall double stranded RNA that regulates the expression of targetmessenger RNAs either by mRNA cleavage, translationalrepression/inhibition or heterochromatic silencing (see for exampleAmbros, 2004, Nature, 431, 350-355; Bartel, 2004, Cell, 116, 281-297;Cullen, 2004, Virus Research., 102, 3-9; He et al., 2004, Nat. Rev.Genet., 5, 522-531; and Ying et al., 2004, Gene, 342, 25-28). In oneembodiment, the microRNA of the invention, has partial complementarity(i.e., less than 100% complementarity) between the sense strand or senseregion and the antisense strand or antisense region of the miRNAmolecule or between the antisense strand or antisense region of themiRNA and a corresponding target nucleic acid molecule. For example,partial complementarity can include various mismatches or non-basepaired nucleotides (e.g., 1, 2, 3, 4, 5 or more mismatches or non-basedpaired nucleotides, such as nucleotide bulges) within the doublestranded nucleic acid molecule, structure which can result in bulges,loops, or overhangs that result between the sense strand or sense regionand the antisense strand or antisense region of the miRNA or between theantisense strand or antisense region of the miRNA and a correspondingtarget nucleic acid molecule.

In one embodiment, compositions of the invention such as formulation orcompositions and formulated siNA compositions of the invention that downregulate or reduce target gene expression are used for preventing ortreating diseases, disorders, conditions, or traits in a subject ororganism as described herein or otherwise known in the art.

By “proliferative disease” or “cancer” as used herein is meant, anydisease, condition, trait, genotype or phenotype characterized byunregulated cell growth or replication as is known in the art; includingleukemias, for example, acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), acute lymphocytic leukemia (ALL), andchronic lymphocytic leukemia, AIDS related cancers such as Kaposi'ssarcoma; breast cancers; bone cancers such as Osteosarcoma,Chondrosarcomas, Ewing's sarcoma, Fibrosarcomas, Giant cell tumors,Adamantinomas, and Chordomas; Brain cancers such as Meningiomas,Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, PituitaryTumors, Schwannomas, and Metastatic brain cancers; cancers of the headand neck including various lymphomas such as mantle cell lymphoma,non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngealcarcinoma, gallbladder and bile duct cancers, cancers of the retina suchas retinoblastoma, cancers of the esophagus, gastric cancers, multiplemyeloma, ovarian cancer, uterine cancer, thyroid cancer, testicularcancer, endometrial cancer, melanoma, colorectal cancer, lung cancer,bladder cancer, prostate cancer, lung cancer (including non-small celllung carcinoma), pancreatic cancer, sarcomas, Wilms' tumor, cervicalcancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma,liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladderadeno carcinoma, parotid adenocarcinoma, endometrial sarcoma, multidrugresistant cancers; and proliferative diseases and conditions, such asneovascularization associated with tumor angiogenesis, maculardegeneration (e.g., wet/dry AMD), corneal neovascularization, diabeticretinopathy, neovascular glaucoma, myopic degeneration and otherproliferative diseases and conditions such as restenosis and polycystickidney disease, and any other cancer or proliferative disease,condition, trait, genotype or phenotype that can respond to themodulation of disease related gene expression in a cell or tissue, aloneor in combination with other therapies.

By “inflammatory disease” or “inflammatory condition” as used herein ismeant any disease, condition, trait, genotype or phenotype characterizedby an inflammatory or allergic process as is known in the art, such asinflammation, acute inflammation, chronic inflammation, respiratorydisease, atherosclerosis, psoriasis, dermatitis, restenosis, asthma,allergic rhinitis, atopic dermatitis, septic shock, rheumatoidarthritis, inflammatory bowl disease, inflammatory pelvic disease, pain,ocular inflammatory disease, celiac disease, Leigh Syndrome, GlycerolKinase Deficiency, Familial eosinophilia (FE), autosomal recessivespastic ataxia, laryngeal inflammatory disease; Tuberculosis, Chroniccholecystitis, Bronchiectasis, Silicosis and other pneumoconioses, andany other inflammatory disease, condition, trait, genotype or phenotypethat can respond to the modulation of disease related gene expression ina cell or tissue, alone or in combination with other therapies.

By “autoimmune disease” or “autoimmune condition” as used herein ismeant, any disease, condition, trait, genotype or phenotypecharacterized by autoimmunity as is known in the art, such as multiplesclerosis, diabetes mellitus, lupus, celiac disease, Crohn's disease,ulcerative colitis, Guillain-Barre syndrome, scleroderms, Goodpasture'ssyndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen'sencephalitis, Primary biliary sclerosis, Sclerosing cholangitis,Autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis,Fibromyalgia, Menier's syndrome; transplantation rejection (e.g.,prevention of allograft rejection) pernicious anemia, rheumatoidarthritis, systemic lupus erythematosus, dermatomyositis, Sjogren'ssyndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis,Reiter's syndrome, Grave's disease, and any other autoimmune disease,condition, trait, genotype or phenotype that can respond to themodulation of disease related gene expression in a cell or tissue, aloneor in combination with other therapies.

By “infectious disease” is meant any disease, condition, trait, genotypeor phenotype associated with an infectious agent, such as a virus,bacteria, fungus, prion, or parasite. Non-limiting examples of variousviral genes that can be targeted using siNA molecules of the inventioninclude Hepatitis C Virus (HCV, for example Genbank Accession Nos:D11168, D50483.1, L38318 and S82227), Hepatitis B Virus (HBV, forexample GenBank Accession No. AF100308.1), Human Immunodeficiency Virustype 1 (HIV-1, for example GenBank Accession No. U51188), HumanImmunodeficiency Virus type 2 (HIV-2, for example GenBank Accession No.X60667), West Nile Virus (WNV for example GenBank accession No.NC_(—)001563), cytomegalovirus (CMV for example GenBank Accession No.NC-001347), respiratory syncytial virus (RSV for example GenBankAccession No. NC_(—)001781), influenza virus (for example GenBankAccession No. AF037412, rhinovirus (for example, GenBank accessionnumbers: D00239, X02316, X01087, L24917, M16248, K02121, X01087),papillomavirus (for example GenBank Accession No. NC_(—)001353), HerpesSimplex Virus (HSV for example GenBank Accession No. NC_(—)001345), andother viruses such as HTLV (for example GenBank Accession No.AJ430-458). Due to the high sequence variability of many viral genomes,selection of siNA molecules for broad therapeutic applications wouldlikely involve the conserved regions of the viral genome. Nonlimitingexamples of conserved regions of the viral genomes include but are notlimited to 5′-Non Coding Regions (NCR), 3′-Non Coding Regions (NCR)and/or internal ribosome entry sites (IRES). siNA molecules designedagainst conserved regions of various viral genomes will enable efficientinhibition of viral replication in diverse patient populations and mayensure the effectiveness of the siNA molecules against viral quasispecies which evolve due to mutations in the non-conserved regions ofthe viral genome. Non-limiting examples of bacterial infections includeActinomycosis, Anthrax, Aspergillosis, Bacteremia, Bacterial Infectionsand Mycoses, Bartonella Infections, Botulism, Brucellosis, BurkholderiaInfections, Campylobacter Infections, Candidiasis, Cat-Scratch Disease,Chlamydia Infections, Cholera, Clostridium Infections,Coccidioidomycosis, Cross Infection, Cryptococcosis, Dermatomycoses,Dermatomycoses, Diphtheria, Ehrlichiosis, Escherichia coli Infections,Fasciitis, Necrotizing, Fusobacterium Infections, Gas Gangrene,Gram-Negative Bacterial Infections, Gram-Positive Bacterial Infections,Histoplasmosis, Impetigo, Klebsiella Infections, Legionellosis, Leprosy,Leptospirosis, Listeria Infections, Lyme Disease, Maduromycosis,Melioidosis, Mycobacterium Infections, Mycoplasma Infections, Mycoses,Nocardia Infections, Onychomycosis, Ornithosis, Plague, PneumococcalInfections, Pseudomonas Infections, Q Fever, Rat-Bite Fever, RelapsingFever, Rheumatic Fever, Rickettsia Infections, Rocky Mountain SpottedFever, Salmonella Infections, Scarlet Fever, Scrub Typhus, Sepsis,Sexually Transmitted Diseases—Bacterial, Bacterial Skin Diseases,Staphylococcal Infections, Streptococcal Infections, Tetanus, Tick-BorneDiseases, Tuberculosis, Tularemia, Typhoid Fever, Typhus, EpidemicLouse-Borne, Vibrio Infections, Yaws, Yersinia Infections, Zoonoses, andZygomycosis. Non-limiting examples of fungal infections includeAspergillosis, Blastomycosis, Coccidioidomycosis, Cryptococcosis, FungalInfections of Fingernails and Toenails, Fungal Sinusitis,Histoplasmosis, Histoplasmosis, Mucormycosis, Nail Fungal Infection,Paracoccidioidomycosis, Sporotrichosis, Valley Fever(Coccidioidomycosis), and Mold Allergy.

By “neurologic disease” or “neurological disease” is meant any disease,disorder, or condition affecting the central or peripheral nervoussystem, including ADHD, AIDS—Neurological Complications, Absence of theSeptum Pellucidum, Acquired Epileptiform Aphasia, Acute DisseminatedEncephalomyelitis, Adrenoleukodystrophy, Agenesis of the CorpusCallosum, Agnosia, Aicardi Syndrome, Alexander Disease, Alpers' Disease,Alternating Hemiplegia, Alzheimer's Disease, Amyotrophic LateralSclerosis, Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis,Anoxia, Aphasia, Apraxia, Arachnoid Cysts, Arachnoiditis, Arnold-ChiariMalformation, Arteriovenous Malformation, Aspartame, Asperger Syndrome,Ataxia Telangiectasia, Ataxia, Attention Deficit-Hyperactivity Disorder,Autism, Autonomic Dysfunction, Back Pain, Barth Syndrome, BattenDisease, Behcet's Disease, Bell's Palsy, Benign Essential Blepharospasm,Benign Focal Amyotrophy, Benign Intracranial Hypertension,Bernhardt-Roth Syndrome, Binswanger's Disease, Blepharospasm,Bloch-Sulzberger Syndrome, Brachial Plexus Birth Injuries, BrachialPlexus Injuries, Bradbury-Eggleston Syndrome, Brain Aneurysm, BrainInjury, Brain and Spinal Tumors, Brown-Sequard Syndrome, BulbospinalMuscular Atrophy, Canavan Disease, Carpal Tunnel Syndrome, Causalgia,Cavemomas, Cavernous Angioma, Cavernous Malformation, Central CervicalCord Syndrome, Central Cord Syndrome, Central Pain Syndrome, CephalicDisorders, Cerebellar Degeneration, Cerebellar Hypoplasia, CerebralAneurysm, Cerebral Arteriosclerosis, Cerebral Atrophy, CerebralBeriberi, Cerebral Gigantism, Cerebral Hypoxia, Cerebral Palsy,Cerebro-Oculo-Facio-Skeletal Syndrome, Charcot-Marie-Tooth Disorder,Chiari Malformation, Chorea, Choreoacanthocytosis, Chronic InflammatoryDemyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance,Chronic Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome, Coma,including Persistent Vegetative State, Complex Regional Pain Syndrome,Congenital Facial Diplegia, Congenital Myasthenia, Congenital Myopathy,Congenital Vascular Cavernous Malformations, Corticobasal Degeneration,Cranial Arteritis, Craniosynostosis, Creutzfeldt-Jakob Disease,Cumulative Trauma Disorders, Cushing's Syndrome, Cytomegalic InclusionBody Disease (CIBD), Cytomegalovirus Infection, Dancing Eyes-DancingFeet Syndrome, Dandy-Walker Syndrome, Dawson Disease, De Morsier'sSyndrome, Dejerine-Klumpke Palsy, Dementia—Multi-Infarct,Dementia—Subcortical, Dementia With Lewy Bodies, Dermatomyositis,Developmental Dyspraxia, Devic's Syndrome, Diabetic Neuropathy, DiffuseSclerosis, Dravet's Syndrome, Dysautonomia, Dysgraphia, Dyslexia,Dysphagia, Dyspraxia, Dystonias, Early Infantile EpilepticEncephalopathy, Empty Sella Syndrome, Encephalitis Lethargica,Encephalitis and Meningitis, Encephaloceles, Encephalopathy,Encephalotrigeminal Angiomatosis, Epilepsy, Erb's Palsy, Erb-Duchenneand Dejerine-Klumpke Palsies, Fabry's Disease, Fahr's Syndrome,Fainting, Familial Dysautonomia, Familial Hemangioma, FamilialIdiopathic Basal Ganglia Calcification, Familial Spastic Paralysis,Febrile Seizures (e.g., GEFS and GEFS plus), Fisher Syndrome, FloppyInfant Syndrome, Friedreich's Ataxia, Gaucher's Disease, Gerstmann'sSyndrome, Gerstmann-Straussler-Scheinker Disease, Giant Cell Arteritis,Giant Cell Inclusion Disease, Globoid Cell Leukodystrophy,Glossopharyngeal Neuralgia, Guillain-Barre Syndrome, HTLV-1 AssociatedMyelopathy, Hallervorden-Spatz Disease, Head Injury, Headache,Hemicrania Continua, Hemifacial Spasm, Hemiplegia Alterans, HereditaryNeuropathies, Hereditary Spastic Paraplegia, Heredopathia AtacticaPolyneuritiformis, Herpes Zoster Oticus, Herpes Zoster, HirayamaSyndrome, Holoprosencephaly, Huntington's Disease, Hydranencephaly,Hydrocephalus—Normal Pressure, Hydrocephalus, Hydromyelia,Hypercortisolism, Hypersomnia, Hypertonia, Hypotonia, Hypoxia,Immune-Mediated Encephalomyelitis, Inclusion Body Myositis,Incontinentia Pigmenti, Infantile Hypotonia, Infantile Phytanic AcidStorage Disease, Infantile Refsum Disease, Infantile Spasms,Inflammatory Myopathy, Intestinal Lipodystrophy, Intracranial Cysts,Intracranial Hypertension, Isaac's Syndrome, Joubert Syndrome,Keams-Sayre Syndrome, Kennedy's Disease, Kinsbourne syndrome,Kleine-Levin syndrome, Klippel Feil Syndrome, Klippel-Trenaunay Syndrome(KTS), Klüver-Bucy Syndrome, Korsakoff's Amnesic Syndrome, KrabbeDisease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton MyasthenicSyndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous NerveEntrapment, Lateral Medullary Syndrome, Learning Disabilities, Leigh'sDisease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy,Levine-Critchley Syndrome, Lewy Body Dementia, Lissencephaly, Locked-InSyndrome, Lou Gehrig's Disease, Lupus—Neurological Sequelae, LymeDisease—Neurological Complications, Machado-Joseph Disease,Macrencephaly, Megalencephaly, Melkersson-Rosenthal Syndrome,Meningitis, Menkes Disease, Meralgia Paresthetica, MetachromaticLeukodystrophy, Microcephaly, Migraine, Miller Fisher Syndrome,Mini-Strokes, Mitochondrial Myopathies, Mobius Syndrome, MonomelicAmyotrophy, Motor Neuron Diseases, Moyamoya Disease, Mucolipidoses,Mucopolysaccharidoses, Multi-Infarct Dementia, Multifocal MotorNeuropathy, Multiple Sclerosis, Multiple System Atrophy with OrthostaticHypotension, Multiple System Atrophy, Muscular Dystrophy,Myasthenia—Congenital, Myasthenia Gravis, Myelinoclastic DiffuseSclerosis, Myoclonic Encephalopathy of Infants, Myoclonus,Myopathy—Congenital, Myopathy—Thyrotoxic, Myopathy, Myotonia Congenita,Myotonia, Narcolepsy, Neuroacanthocytosis, Neurodegeneration with BrainIron Accumulation, Neurofibromatosis, Neuroleptic Malignant Syndrome,Neurological Complications of AIDS, Neurological Manifestations of PompeDisease, Neuromyelitis Optica, Neuromyotonia, Neuronal CeroidLipofuscinosis, Neuronal Migration Disorders, Neuropathy—Hereditary,Neurosarcoidosis, Neurotoxicity, Nevus Cavernosus, Niemann-Pick Disease,O'Sullivan-McLeod Syndrome, Occipital Neuralgia, Occult SpinalDysraphism Sequence, Ohtahara Syndrome, Olivopontocerebellar Atrophy,Opsoclonus Myoclonus, Orthostatic Hypotension, Overuse Syndrome,Pain—Chronic, Paraneoplastic Syndromes, Paresthesia, Parkinson'sDisease, Parmyotonia Congenita, Paroxysmal Choreoathetosis, ParoxysmalHemicrania, Parry-Romberg, Pelizaeus-Merzbacher Disease, Pena Shokeir IISyndrome, Perineural Cysts, Periodic Paralyses, Peripheral Neuropathy,Periventricular Leukomalacia, Persistent Vegetative State, PervasiveDevelopmental Disorders, Phytanic Acid Storage Disease, Pick's Disease,Piriformis Syndrome, Pituitary Tumors, Polymyositis, Pompe Disease,Porencephaly, Post-Polio Syndrome, Postherpetic Neuralgia,Postinfectious Encephalomyelitis, Postural Hypotension, PosturalOrthostatic Tachycardia Syndrome, Postural Tachycardia Syndrome, PrimaryLateral Sclerosis, Prion Diseases, Progressive Hemifacial Atrophy,Progressive Locomotor Ataxia, Progressive MultifocalLeukoencephalopathy, Progressive Sclerosing Poliodystrophy, ProgressiveSupranuclear Palsy, Pseudotumor Cerebri, Pyridoxine Dependent andPyridoxine Responsive Siezure Disorders, Ramsay Hunt Syndrome Type I,Ramsay Hunt Syndrome Type II, Rasmussen's Encephalitis and otherautoimmune epilepsies, Reflex Sympathetic Dystrophy Syndrome, RefsumDisease—Infantile, Refsum Disease, Repetitive Motion Disorders,Repetitive Stress Injuries, Restless Legs Syndrome,Retrovirus-Associated Myelopathy, Rett Syndrome, Reye's Syndrome,Riley-Day Syndrome, SUNCT Headache, Sacral Nerve Root Cysts, Saint VitusDance, Salivary Gland Disease, Sandhoff Disease, Schilder's Disease,Schizencephaly, Seizure Disorders, Septo-Optic Dysplasia, SevereMyoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome, Shingles,Shy-Drager Syndrome, Sjogren's Syndrome, Sleep Apnea, Sleeping Sickness,Soto's Syndrome, Spasticity, Spina Bifida, Spinal Cord Infarction,Spinal Cord Injury, Spinal Cord Tumors, Spinal Muscular Atrophy,Spinocerebellar Atrophy, Steele-Richardson-Olszewski Syndrome,Stiff-Person Syndrome, Striatonigral Degeneration, Stroke, Sturge-WeberSyndrome, Subacute Sclerosing Panencephalitis, SubcorticalArteriosclerotic Encephalopathy, Swallowing Disorders, Sydenham Chorea,Syncope, Syphilitic Spinal Sclerosis, Syringohydromyelia, Syringomyelia,Systemic Lupus Erythematosus, Tabes Dorsalis, Tardive Dyskinesia, TarlovCysts, Tay-Sachs Disease, Temporal Arteritis, Tethered Spinal CordSyndrome, Thomsen Disease, Thoracic Outlet Syndrome, ThyrotoxicMyopathy, Tic Douloureux, Todd's Paralysis, Tourette Syndrome, TransientIschemic Attack, Transmissible Spongiform Encephalopathies, TransverseMyelitis, Traumatic Brain Injury, Tremor, Trigeminal Neuralgia, TropicalSpastic Paraparesis, Tuberous Sclerosis, Vascular Erectile Tumor,Vasculitis including Temporal Arteritis, Von Economo's Disease, VonHippel-Lindau disease (VHL), Von Recklinghausen's Disease, Wallenberg'sSyndrome, Werdnig-Hoffman Disease, Wemicke-Korsakoff Syndrome, WestSyndrome, Whipple's Disease, Williams Syndrome, Wilson's Disease,X-Linked Spinal and Bulbar Muscular Atrophy, and Zellweger Syndrome.

By “respiratory disease” is meant, any disease or condition affectingthe respiratory tract, such as asthma, chronic obstructive pulmonarydisease or “COPD”, allergic rhinitis, sinusitis, pulmonaryvasoconstriction, inflammation, allergies, impeded respiration,respiratory distress syndrome, cystic fibrosis, pulmonary hypertension,pulmonary vasoconstriction, emphysema, and any other respiratorydisease, condition, trait, genotype or phenotype that can respond to themodulation of disease related gene expression in a cell or tissue, aloneor in combination with other therapies.

By “cardiovascular disease” is meant and disease or condition affectingthe heart and vasculature, including but not limited to, coronary heartdisease (CHD), cerebrovascular disease (CVD), aortic stenosis,peripheral vascular disease, atherosclerosis, arteriosclerosis,myocardial infarction (heart attack), cerebrovascular diseases (stroke),transient ischaemic attacks (TIA), angina (stable and unstable), atrialfibrillation, arrhythmia, vavular disease, congestive heart failure,hypercholoesterolemia, type I hyperlipoproteinemia, type IIhyperlipoproteinemia, type III hyperlipoproteinemia, type IVhyperlipoproteinemia, type V hyperlipoproteinemia, secondaryhypertrigliceridemia, and familial lecithin cholesterol acyltransferasedeficiency.

By “ocular disease” as used herein is meant, any disease, condition,trait, genotype or phenotype of the eye and related structures as isknown in the art, such as Cystoid Macular Edema, Asteroid Hyalosis,Pathological Myopia and Posterior Staphyloma, Toxocariasis (Ocular LarvaMigrans), Retinal Vein Occlusion, Posterior Vitreous Detachment,Tractional Retinal Tears, Epiretinal Membrane, Diabetic Retinopathy,Lattice Degeneration, Retinal Vein Occlusion, Retinal Artery Occlusion,Macular Degeneration (e.g., age related macular degeneration such as wetAMD or dry AMD), Toxoplasmosis, Choroidal Melanoma, AcquiredRetinoschisis, Hollenhorst Plaque, Idiopathic Central SerousChorioretinopathy, Macular Hole, Presumed Ocular HistoplasmosisSyndrome, Retinal Macroaneursym, Retinitis Pigmentosa, RetinalDetachment, Hypertensive Retinopathy, Retinal Pigment Epithelium (RPE)Detachment, Papillophlebitis, Ocular Ischemic Syndrome, Coats' Disease,Leber's Miliary Aneurysm, Conjunctival Neoplasms, AllergicConjunctivitis, Vernal Conjunctivitis, Acute Bacterial Conjunctivitis,Allergic Conjunctivitis &Vernal Keratoconjunctivitis, ViralConjunctivitis, Bacterial Conjunctivitis, Chlamydial & GonococcalConjunctivitis, Conjunctival Laceration, Episcleritis, Scleritis,Pingueculitis, Pterygium, Superior Limbic Keratoconjunctivitis (SLK ofTheodore), Toxic Conjunctivitis, Conjunctivitis with Pseudomembrane,Giant Papillary Conjunctivitis, Terrien's Marginal Degeneration,Acanthamoeba Keratitis, Fungal Keratitis, Filamentary Keratitis,Bacterial Keratitis, Keratitis Sicca/Dry Eye Syndrome, BacterialKeratitis, Herpes Simplex Keratitis, Sterile Corneal Infiltrates,Phlyctenulosis, Corneal Abrasion & Recurrent Corneal Erosion, CornealForeign Body, Chemical Burs, Epithelial Basement Membrane Dystrophy(EBMD), Thygeson's Superficial Punctate Keratopathy, Corneal Laceration,Salzmann's Nodular Degeneration, Fuchs' Endothelial Dystrophy,Crystalline Lens Subluxation, Ciliary-Block Glaucoma, Primary Open-AngleGlaucoma, Pigment Dispersion Syndrome and Pigmentary Glaucoma,Pseudoexfoliation Syndrom and Pseudoexfoliative Glaucoma, AnteriorUveitis, Primary Open Angle Glaucoma, Uveitic Glaucoma &Glaucomatocyclitic Crisis, Pigment Dispersion Syndrome & PigmentaryGlaucoma, Acute Angle Closure Glaucoma, Anterior Uveitis, Hyphema, AngleRecession Glaucoma, Lens Induced Glaucoma, Pseudoexfoliation Syndromeand Pseudoexfoliative Glaucoma, Axenfeld-Rieger Syndrome, NeovascularGlaucoma, Pars Planitis, Choroidal Rupture, Duane's Retraction Syndrome,Toxic/Nutritional Optic Neuropathy, Aberrant Regeneration of CranialNerve III, Intracranial Mass Lesions, Carotid-Cavernous Sinus Fistula,Anterior Ischemic Optic Neuropathy, Optic Disc Edema & Papilledema,Cranial Nerve III Palsy, Cranial Nerve IV Palsy, Cranial Nerve VI Palsy,Cranial Nerve VII (Facial Nerve) Palsy, Horner's Syndrome, InternuclearOpthalmoplegia, Optic Nerve Head Hypoplasia, Optic Pit, Tonic Pupil,Optic Nerve Head Drusen, Demyelinating Optic Neuropathy (Optic Neuritis,Retrobulbar Optic Neuritis), Amaurosis Fugax and Transient IschemicAttack, Pseudotumor Cerebri, Pituitary Adenoma, Molluscum Contagiosum,Canaliculitis, Verruca and Papilloma, Pediculosis and Pthiriasis,Blepharitis, Hordeolum, Preseptal Cellulitis, Chalazion, Basal CellCarcinoma, Herpes Zoster Ophthalmicus, Pediculosis & Phthiriasis,Blow-out Fracture, Chronic Epiphora, Dacryocystitis, Herpes SimplexBlepharitis, Orbital Cellulitis, Senile Entropion, and Squamous CellCarcinoma.

By “metabolic disease” is meant any disease or condition affectingmetabolic pathways as in known in the art. Metabolic disease can resultin an abnormal metabolic process, either congenital due to inheritedenzyme abnormality (inborn errors of metabolism) or acquired due todisease of an endocrine organ or failure of a metabolically importantorgan such as the liver. In one embodiment, metabolic disease includesobesity, insulin resistance, and diabetes (e.g., type I and/or type IIdiabetes).

By “dermatological disease” is meant any disease or condition of theskin, dermis, or any substructure therein such as hair, follicle, etc.Dermatological diseases, disorders, conditions, and traits can includepsoriasis, ectopic dermatitis, skin cancers such as melanoma and basalcell carcinoma, hair loss, hair removal, alterations in pigmentation,and any other disease, condition, or trait associated with the skin,dermis, or structures therein.

By “auditory disease” is meant any disease or condition of the auditorysystem, including the ear, such as the inner ear, middle ear, outer ear,auditory nerve, and any substructures therein. Auditory diseases,disorders, conditions, and traits can include hearing loss, deafness,tinnitus, Meniere's Disease, vertigo, balance and motion disorders, andany other disease, condition, or trait associated with the ear, orstructures therein.

In one embodiment of the present invention, each sequence of a siNAmolecule of the invention is independently about 15 to about 30nucleotides in length, in specific embodiments about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. Inanother embodiment, the siNA duplexes of the invention independentlycomprise about 15 to about 30 base pairs (e.g., about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In anotherembodiment, one or more strands of the siNA molecule of the inventionindependently comprises about 15 to about 30 nucleotides (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) thatare complementary to a target nucleic acid molecule. In yet anotherembodiment, siNA molecules of the invention comprising hairpin orcircular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50or 55) nucleotides in length, or about 38 to about 44 (e.g., about 38,39, 40, 41, 42, 43, or 44) nucleotides in length and comprising about 15to about 25 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25)base pairs.

As used herein “cell” is used in its usual biological sense, and doesnot refer to an entire multicellular organism, e.g., specifically doesnot refer to a human. The cell can be present in an organism, e.g.,birds, plants and mammals such as humans, cows, sheep, apes, monkeys,swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterialcell) or eukaryotic (e.g., mammalian or plant cell). The cell can be ofsomatic or germ line origin, totipotent or pluripotent, dividing ornon-dividing. The cell can also be derived from or can comprise a gameteor embryo, a stem cell, or a fully differentiated cell.

In one embodiment, a formulation or composition or formulated siNAcomposition of the invention is locally administered to relevant tissuesex vivo, or in vivo through direct injection, catheterization, orstenting (e.g., portal vein catherization/stenting).

In one embodiment, a formulation or composition or formulated siNAcomposition of the invention is systemically delivered to a subject ororganism through parental administration as is known in the art, such asvia intravenous, intramuscular, or subcutaneous injection.

In another aspect, the invention provides mammalian cells containing oneor more formulation or composition or formulated siNA compositions ofthis invention. The one or more formulation or composition or formulatedsiNA compositions can independently be targeted to the same or differentsites.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a β-D-ribofuranose moiety. The terms includedouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of the invention can beadministered. A subject can be a mammal or mammalian cells, including ahuman or human cells.

The term “phosphorothioate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise a sulfur atom.Hence, the term phosphorothioate refers to both phosphorothioate andphosphorodithioate internucleotide linkages.

The term “phosphonoacetate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise an acetyl orprotected acetyl group.

The term “thiophosphonoacetate” as used herein refers to aninternucleotide linkage having Formula I, wherein Z comprises an acetylor protected acetyl group and W comprises a sulfur atom or alternately Wcomprises an acetyl or protected acetyl group and Z comprises a sulfuratom.

The term “universal base” as used herein refers to nucleotide baseanalogs that form base pairs with each of the natural DNA/RNA bases withlittle discrimination between them. Non-limiting examples of universalbases include C-phenyl, C-naphthyl and other aromatic derivatives,inosine, azole carboxamides, and nitroazole derivatives such as3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as knownin the art (see for example Loakes, 2001, Nucleic Acids Research, 29,2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotidehaving an acyclic ribose sugar, for example where any of the ribosecarbons (C1, C2, C3, C4, or C5), are independently or in combinationabsent from the nucleotide.

In a further embodiment, the formulation or compositions and formulatedsiNA compositions can be used in combination with other known treatmentsto inhibit, reduce, or prevent diseases, traits, and conditionsdescribed herein or otherwise known in the art in a subject or organism.For example, the described molecules could be used in combination withone or more known compounds, treatments, or procedures to inhibit,reduce, or prevent diseases, traits, and conditions described herein orotherwise known in the art in a subject or organism. In a non-limitingexample, formulation or composition and formulated siNA compositionsthat are used to treat HCV infection and comorbid conditions that areassociated with HBV infection are used in combination with other HCVtreatments, such as HCV vaccines; anti-HCV antibodies such as HepeX-Cand Civacir; protease inhibitors such as VX-950; pegylated interferonssuch as PEG-Intron, and/or other antivirals such as Ribavirin and/orValopicitabine.

In one embodiment, a formulated siNA composition of the inventioncomprises an expression vector comprising a nucleic acid sequenceencoding at least one polynucleotide molecule of the invention (e.g.,siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule) in amanner which allows expression of the siNA molecule. For example, thevector can contain sequence(s) encoding both strands of a siNA moleculecomprising a duplex. The vector can also contain sequence(s) encoding asingle nucleic acid molecule that is self-complementary and thus forms asiNA molecule. Non-limiting examples of such expression vectors aredescribed in Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishiand Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, NatureBiotechnology, 19, 500; and Novina et al., 2002, Nature Medicine,advance online publication doi:10.1038/nm725. In one embodiment, anexpression vector of the invention comprises a nucleic acid sequenceencoding two or more siNA molecules, which can be the same or different.

In another aspect of the invention, polynucleotides of the inventionsuch as siNA molecules that interact with target RNA molecules anddown-regulate gene encoding target RNA molecules (for example target RNAmolecules referred to by Genbank Accession numbers herein) are expressedfrom transcription units inserted into DNA or RNA vectors. Therecombinant vectors can be DNA plasmids or viral vectors. Polynucleotideexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Therecombinant vectors capable of expressing the polynucleotide moleculescan be delivered as described herein, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of polynucleotide molecules. Such vectors can be repeatedlyadministered as necessary. For example, once expressed, the siNAmolecules bind and down-regulate gene function or expression via RNAinterference (RNAi). Delivery of formulation or composition s expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from asubject followed by reintroduction into the subject, or by any othermeans that would allow for introduction into the desired target cell.

By “vectors” is meant any nucleic acid- and/or viral-based techniqueused to deliver a desired nucleic acid.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non limiting example of a composition comprising a firstvehicle including one or more biologically active molecules (B), and asecond vehicle including one or more carrier molecules (X), for exampleas a heterogeneous population. The first vehicle and the second vehiclecan the same with the exception of the biologically active molecule(s)and the carrier molecule(s) (designated Formulation Type A1, see FIG.1A). The first vehicle and the second vehicle can also be different(designated Formulation Type A2, see FIG. 1B). The first vehicle and thesecond vehicle can be present in equal ratios (e.g., 1:1) or indiffering ratios.

FIG. 2 shows a non limiting example of a composition comprising avehicle including one or more biologically active molecules (B) and oneor more carrier molecules (X), for example as a homogeneous population(designated Formulation Type B). The biologically active molecule (B)and the carrier molecule (X) can be present in equal ratios (e.g., 1:1)or in differing ratios.

FIG. 3 shows a non limiting example of a composition comprising one ormore carrier molecules (X), and a vehicle including one or morebiologically active molecules (B), for example as a heterogeneouspopulation (designated Formulation Type C). The vehicle and the carriermolecule (X) can be present in equal ratios (e.g., 1:1) or in differingratios.

FIG. 4 shows a non limiting example of a composition comprising a firstformulation including one or more carrier molecules (X) and a secondformulation including one or more biologically active molecules (B)(e.g., a polynucleotide such as a siNA, miRNA, RNAi inhibitor,antisense, aptamer, decoy, ribozyme, 2-5A, triplex formingoligonucleotide, other nucleic acid molecule and/or other biologicallyactive molecule described herein), a cationic lipid, a neutral lipid,and a polyethyleneglycol conjugate, such as a PEG-diacylglycerol,PEG-diacylglycamide, PEG-cholesterol, or PEG-DMB conjugate. The firstand/or second formulation can further comprise cholesterol or acholesterol derivative. The first and/or second formulation can furthercomprise an alcohol or surfactant. The first and/or second formulationcan further comprise lineoyl alcohol. This composition is generallyreferred to herein as LNP Formulation Type A. The first formulation andthe second formulation can be present in equal ratios (e.g., 1:1) or indiffering ratios.

FIG. 5 shows a non limiting example of a composition comprising aformulation including one or more carrier molecules (X), one or morebiologically active molecules (B) (e.g., a polynucleotide such as asiNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, other nucleic acid molecule and/orother biologically active molecule described herein), a cationic lipid,a neutral lipid, and a polyethyleneglycol conjugate, such as aPEG-diacylglycerol, PEG-diacylglycamide, PEG-cholesterol, or PEG-DMBconjugate. The formulation can further comprise cholesterol or acholesterol derivative. The formulation can further comprise an alcoholor surfactant. The formulation can further comprise lineoyl alcohol.This composition is generally referred to herein as LNP Formulation TypeB. The biologically active molecule (B) and the carrier molecule (X) canbe present in equal ratios (e.g., 1:1) or in differing ratios.

FIG. 6 shows a non limiting example of a composition comprising one ormore carrier molecules (X), and a formulation including one or morebiologically active molecules (B) (e.g., a polynucleotide such as asiNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, other nucleic acid molecule and/orother biologically active molecule described herein), a cationic lipid,a neutral lipid, and a polyethyleneglycol conjugate, such as aPEG-diacylglycerol, PEG-diacylglycamide, PEG-cholesterol, or PEG-DMBconjugate. The formulation can further comprise cholesterol or acholesterol derivative. The formulation can further comprise an alcoholor surfactant. The formulation can further comprise lineoyl alcohol.This composition is generally referred to herein as LNP Formulation TypeC. The vehicle and the carrier molecule (X) can be present in equalratios (e.g., 1:1) or in differing ratios.

FIG. 7 shows non-limiting examples of cationic lipid compounds of theinvention.

FIG. 8 shows non-limiting examples of acetal linked cationic lipidcompounds of the invention.

FIG. 9 shows non-limiting examples of succinyl/acyl linked cationiclipid compounds of the invention.

FIG. 10 shows non-limiting examples of aromatic cationic lipid compoundsof the invention.

FIG. 11 shows non-limiting examples of additional cationic lipidcompounds of the invention.

FIG. 12 shows a schematic of the components of a formulation orcomposition.

FIG. 13 shows a schematic diagram of the lamellar structure and invertedhexagonal structure that can be adopted by a formulation or composition.

FIG. 14 shows the components of L051, a serum-stable formulation orcomposition that undergoes a rapid pH-dependent phase transition.

FIG. 15 shows the components of L073, a serum-stable formulation orcomposition that undergoes a rapid pH-dependent phase transition.

FIG. 16 shows the components of L069, a serum-stable formulation orcomposition that undergoes a rapid pH-dependent phase transition.

FIG. 17 shows a graph depicting the serum stability of formulation orcomposition s L065, F2, L051, and L073 as determined by the relativeturbidity of the formulation or composition s in 50% serum measured byabsorbance at 500 nm. Formulation or composition s L065, L051, and L073are stable in serum.

FIG. 18 shows a graph depicting the pH-dependent phase transition offormulation or composition s L065, F2, L051, and L073 as determined bythe relative turbidity of the formulation or composition s in buffersolutions ranging from pH 3.5 to pH 9.0 measured by absorbance at 350nm. Formulation or composition s L051 and L073 each undergo a rapidpH-dependent phase transition at pH 5.5-pH 6.5.

FIG. 19 shows a graph depicting the pH-dependent phase transition offormulation or composition L069 as determined by the relative turbidityof the formulation or composition in buffer solutions ranging from pH3.5 to pH 9.0 measured by absorbance at 350 nm. Formulation orcomposition L069 undergoes a rapid pH-dependent phase transition at pH5.5-pH 6.5.

FIG. 20 shows a non-limiting example of chemical modifications of siNAmolecules of the invention.

FIG. 21 shows a non-limiting example of in vitro efficacy of siNAnanoparticles in reducing HBsAg levels in HepG2 cells. Active chemicallymodified siNA molecules were designed to target HBV site 263 RNA (siNAsequences are shown in FIG. 20). The figure shows the level of HBsAg incells treated with formulated active siNA L051 nanoparticles (see TableIV) compared to untreated or negative control treated cells. A dosedependent reduction in HBsAg levels was observed in the active siNAtreated cells, while no reduction is observed in the negative controltreated cells.

FIG. 22 shows a non-limiting example of in vitro efficacy of siNAnanoparticles in reducing HBsAg levels in HepG2 cells. Active chemicallymodified siNA molecules were designed to target HBV site 263 RNA (siNAsequences are shown in FIG. 20). The figure shows the level of HBsAg incells treated with formulated active siNA L053 and L054 nanoparticles(see Table IV) compared to untreated or negative control treated cells.A dose dependent reduction in HBsAg levels was observed in the activesiNA treated cells, while no reduction is observed in the negativecontrol treated cells.

FIG. 23 shows a non-limiting example of in vitro efficacy of siNAnanoparticles in reducing HBsAg levels in HepG2 cells. Active chemicallymodified siNA molecules were designed to target HBV site 263 RNA (siNAsequences are shown in FIG. 20). The figure shows the level of HBsAg incells treated with formulated molecular composition L069 comprisingactive siNA (see Table IV) compared to untreated or negative controltreated cells. A dose dependent reduction in HBsAg levels was observedin the active siNA treated cells, while no reduction is observed in thenegative control treated cells.

FIG. 24 shows a non-limiting example of the activity of systemicallyadministered siNA L051 (Table IV) nanoparticles in an HBV mouse model. Ahydrodynamic tail vein injection was done containing 0.3 μg of the pWTDHBV vector. The nanoparticle encapsulated active siNA molecules wereadministered at 3 mg/kg/day for three days via standard IV injectionbeginning 6 days post-HDI. Groups (N=5) of animals were sacrificed at 3and 7 days following the last dose, and the levels of serum HBV DNA wasmeasured. HBV DNA titers were determined by quantitative real-time PCRand expressed as mean log 10 copies/ml (±SEM).

FIG. 25 shows a non-limiting example of the activity of systemicallyadministered siNA L051 (Table IV) nanoparticles in an HBV mouse model. Ahydrodynamic tail vein injection was done containing 0.3 μg of the pWTDHBV vector. The nanoparticle encapsulated active siNA molecules wereadministered at 3 mg/kg/day for three days via standard IV injectionbeginning 6 days post-HDI. Groups (N=5) of animals were sacrificed at 3and 7 days following the last dose, and the levels of serum HBsAg wasmeasured. The serum HBsAg levels were assayed by ELISA and expressed asmean log 10 pg/ml (±SEM).

FIG. 26 shows a non-limiting example of formulated siNA L051 (Table IV)nanoparticle constructs targeting viral replication in a Huh7 HCVreplicon system in a dose dependent manner. Active siNA formulationswere evaluated at 1, 5, 10, and 25 nM in comparison to untreated cells(“untreated”), and formulated inactive siNA scrambled control constructsat the same concentration.

FIG. 27 shows a non-limiting example of formulated siNA L053 and L054(Table IV) nanoparticle constructs targeting viral replication in a Huh7HCV replicon system in a dose dependent manner. Active siNA formulationswere evaluated at 1, 5, 10, and 25 nM in comparison to untreated cells(“untreated”), and formulated inactive siNA scrambled control constructsat the same concentration.

FIG. 28 shows the distribution of siNA in lung tissue of mice followingintratracheal dosing of unformulated siNA, cholesterol-conjugated siNA,and formulated siNA (formulated molecular compositions 18.1 and 19.1).As shown, the longest half lives of exposure in lung tissue wereobserved with the siNA formulated in molecular compositions T018.1 orT019.1.

FIG. 29A shows a non-limiting example of a synthetic scheme used for thesynthesis of3-Dimethylamino-2-(Cholest-5-en-3β-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA).

FIG. 29B shows a non-limiting example of an alternative synthetic schemeused for the synthesis of3-Dimethylamino-2-(Cholest-5-en-3β-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA).

FIG. 29C shows a non-limiting example of a synthetic scheme used for thesynthesis of N,N-Dimethyl-3,4-dilinoleyloxybenzylamine andN,N-Dimethyl-3,4-dioleyloxybenzylamine.

FIG. 30A shows a non-limiting example of a synthetic scheme used for thesynthesis of1-[8′-(Cholest-5-en-3β-oxy)carboxamido-3′,6′-dioxaoctanyl]carbamoyl-co-methyl-poly(ethyleneglycol) (PEG-cholesterol) and3,4-Ditetradecoxyylbenzyl-ω-methyl-poly(ethylene glycol)ether (PEG-DMB).In the Figure, PEG is PEG2000, a polydispersion which can typically varyfrom ˜1500 to ˜3000 Da represented by the formula PEG_(n) (i.e., where nis about 33 to about 67, or on average ˜45).

FIG. 30B shows a non-limiting example of a synthetic scheme used for thesynthesis of1-[8′-(1,2-Dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethyleneglycol) (PEG-DMG). In the Figure, PEG is PEG2000, a polydispersion whichcan typically vary from ˜1500 to ˜3000 Da represented by the formulaPEG_(n) (i.e., where n is about 33 to about 67, or on average ˜45).

FIG. 31 shows the components of L083, a serum-stable formulation orcomposition that undergoes a rapid pH-dependent phase transition.

FIG. 32 shows the components of L077, a serum-stable formulation orcomposition that undergoes a rapid pH-dependent phase transition.

FIG. 33 shows the components of L080, a serum-stable formulation orcomposition that undergoes a rapid pH-dependent phase transition.

FIG. 34 shows the components of L082, a serum-stable formulation orcomposition that undergoes a rapid pH-dependent phase transition.

FIG. 35 shows a non-limiting example of the activity of systemicallyadministered siNA L077, L069, L080, L082, L083, L060, L061, and L051(Table IV) nanoparticles in an HBV mouse model. A hydrodynamic tail veininjection was done containing 0.3 μg of the pWTD HBV vector. Thenanoparticle encapsulated active siNA molecules were administered at 3mg/kg/day for three days via standard IV injection beginning 6 dayspost-HDI. Groups (N=5) of animals were sacrificed at 3 and 7 daysfollowing the last dose, and the levels of serum HBV DNA was measured.HBV DNA titers were determined by quantitative real-time PCR andexpressed as mean log 10 copies/ml (±SEM).

FIG. 36 shows a non-limiting example of the dose response activity ofsystemically administered siNA L083 and L084 (Table IV) nanoparticles inan HBV mouse model. A hydrodynamic tail vein injection was donecontaining 0.3 μg of the pWTD HBV vector. The nanoparticle encapsulatedactive siNA molecules were administered at 3 mg/kg/day for three daysvia standard IV injection beginning 6 days post-HDI. Groups (N=5) ofanimals were sacrificed at 3 and 7 days following the last dose, and thelevels of serum HBsAg was measured. The serum HBsAg levels were assayedby ELISA and expressed as mean log 10 pg/ml (±SEM).

FIG. 37 shows a non-limiting example of the dose response activity ofsystemically administered siNA L077 (Table IV) nanoparticles in an HBVmouse model. A hydrodynamic tail vein injection was done containing 0.3μg of the pWTD HBV vector. The nanoparticle encapsulated active siNAmolecules were administered at 3 mg/kg/day for three days via standardIV injection beginning 6 days post-HDI. Groups (N=5) of animals weresacrificed at 3 and 7 days following the last dose, and the levels ofserum HBsAg was measured. The serum HBsAg levels were assayed by ELISAand expressed as mean log 10 μg/ml (±SEM).

FIG. 38 shows a non-limiting example of the dose response activity ofsystemically administered siNA L080 (Table IV) nanoparticles in an HBVmouse model. A hydrodynamic tail vein injection was done containing 0.3μg of the pWTD HBV vector. The nanoparticle encapsulated active siNAmolecules were administered at 3 mg/kg/day for three days via standardIV injection beginning 6 days post-HDI. Groups (N=5) of animals weresacrificed at 3 and 7 days following the last dose, and the levels ofserum HBsAg was measured. The serum HBsAg levels were assayed by ELISAand expressed as mean log 10 μg/ml (±SEM).

FIG. 39 shows a non-limiting example of the serum stability of siNAL077, L080, L082, and L083 (Table IV) nanoparticle formulations.

FIG. 40 shows a graph depicting the pH-dependent phase transition ofsiNA L077, L080, L082, and L083 (Table IV) nanoparticle formulations asdetermined by the relative turbidity of the formulated molecularcomposition in buffer solutions ranging from pH 3.5 to pH 9.0 measuredby absorbance at 350 nm. Formulated molecular composition L069 undergoesa rapid pH-dependent phase transition at pH 5.5-pH 6.5.

FIG. 41 shows efficacy data for LNP 58 and LNP 98 formulations targetingMapK14 site 1033 in RAW 264.7 mouse macrophage cells compared to LFK2000and a formulated irrelevant siNA control.

FIG. 42 shows efficacy data for LNP 98 formulations targeting MapK14site 1033 in MM14.Lu normal mouse lung cells compared to LFK2000 and aformulated irrelevant siNA control.

FIG. 43 shows efficacy data for LNP 54, LNP 97, and LNP 98 formulationstargeting MapK14 site 1033 in 6.12 B lymphocyte cells compared toLFK2000 and a formulated irrelevant siNA control.

FIG. 44 shows efficacy data for LNP 98 formulations targeting MapK14site 1033 in NIH 3T3 cells compared to LFK2000 and a formulatedirrelevant siNA control.

FIG. 45 shows the dose-dependent reduction of MapK14 RNA via MapK14 LNP54 and LNP 98 formulated siNAs in RAW 264.7 cells.

FIG. 46 shows the dose-dependent reduction of MapK14 RNA via MapK14 LNP98 formulated siNAs in MM14.Lu cells.

FIG. 47 shows the dose-dependent reduction of MapK14 RNA via MapK14 LNP97 and LNP 98 formulated siNAs in 6.12 B cells.

FIG. 48 shows the dose-dependent reduction of MapK14 RNA via MapK14 LNP98 formulated siNAs in NIH 3T3 cells.

FIG. 49 shows a non-limiting example of reduced airwayhyper-responsiveness from treatment with LNP-51 formulated siNAstargeting IL-4R in a mouse model of OVA challenge mediated airwayhyper-responsiveness. Active formulated siNAs were tested at 0.01, 0.1,and 1.0 mg/kg and were compared to LNP vehicle along and untreated(naïve) animals.

FIG. 50 shows a non-limiting example of LNP formulated siNA mediatedinhibition of huntingtin (htt) gene expression in vivo. Using Alzetosmotic pumps, siNAs encapsulated in LNPs were infused into mouselateral ventrical or striatum for 7 or 14 days, respectively, atconcentrations ranging from 0.1 to 1 mg/ml (total dose ranging from 8.4to 84 μg). Animals treated with active siNA formulated with LNP-098 orLNP-061 were compared to mismatch control siNA formulated with LNP-061and untreated animal controls. Huntingtin (htt) gene expression levelswere determined by QPCR.

FIG. 51 shows a non-limiting example of the dose dependent anti-HBVactivity of active LNP formulated HBV263 siNA in presence and absence ofa carrier LNP formulation of inactive siNA, compared to an untreatedcontrol.

FIG. 52 shows a non-limiting example of the dose dependent knockdown ofSSB target RNA in mouse liver by active LNP formulated SSB291 siNA inpresence and absence of carrier LNP formulation of inactive siNA,compared to an untreated control.

FIG. 53 shows a non-limiting example of the effect of LNP formulatedsingle strand or duplex polynucleotide carrier molecules on RNAiactivity of active LNP formulated SSB291 siNA, compared to an untreatedcontrol.

FIG. 54 shows a non-limiting example of the activity of the carriereffect. To explore if the carrier effect allows for efficient RNAi formultiple siRNAs in a mixture, siRNAs were used targeting 3 differentgenes. The SSB 291, CRTC2:283 and IKK2 2389 siRNAs were dosed at 3 mg/kgalone or as mixture of all three along with 2.1 mg/kg carrier HCV316 ata total dose of 3 mg/kg. When the siRNAs were dosed individually at 0.3mg/kg, they showed moderate to no knockdown of their intended target. Onthe other hand, when given as mixture, knockdown efficiency was improvedsignificantly. For SSB291, the target knockdown improved from 31% whengiven alone to 77% when given in a mixture. For CRTC2:283, the targetknockdown improved from 17% when given alone to 41% when given in amixture. For IKK1:2389, no target knockdown was observed when givenalone but it improved to 48% when given in a mixture. Thus, even thoughthe concentration of each siRNA was 0.3 mg/kg, when given alone or in amixture, significant improvement in activity was achieved by dosing themas a mixture. This allows targeting of multiple targets at lower dosesto achieve additive or synergistic effects.

FIG. 55 shows a non-limiting example of the use of empty LNP forbeneficial carrier effect. To further understand the nature of carriercargo, empty L201 was prepared. The SSB291 L201 was injected at 1 mg/kgalone, or in the presence of empty L201 as carrier at a total dose of 3mg/kg. The total liver RNA was isolated and analysed for SSB RNA. SSB291showed 54% knockdown of target RNA when dosed alone but when suppliedwith empty LNP carrier, the knockdown efficiency improved to 79%. Thecarrier empty LNP on its own showed no significant knockdown of SSBtarget. This result shows that potentiation of RNAi activity can beachieved by empty LNP, “empty carrier”.

DETAILED DESCRIPTION OF THE INVENTION Mechanism of Action of NucleicAcid Molecules of the Invention

Aptamer: Nucleic acid aptamers can be selected to specifically bind to aparticular ligand of interest (see for example Gold et al., U.S. Pat.No. 5,567,588 and U.S. Pat. No. 5,475,096, Gold et al., 1995, Annu. Rev.Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun,2000, Curr. Opin. Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74,27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999,Clinical Chemistry, 45, 1628). For example, the use of in vitroselection can be applied to evolve nucleic acid aptamers with bindingspecificity for CylA. Nucleic acid aptamers can include chemicalmodifications and linkers as described herein. Nucleic aptamers of theinvention can be double stranded or single stranded and can comprise onedistinct nucleic acid sequence or more than one nucleic acid sequencescomplexed with one another. Aptamer molecules of the invention that bindto CylA, can modulate the protease activity of CylA and subsequentactivation of cytolysin, and therefore modulate the acute toxicityassociated with enterococcal infection.

Antisense: Antisense molecules can be modified or unmodified RNA, DNA,or mixed polymer oligonucleotides and primarily function by specificallybinding to matching sequences resulting in modulation of peptidesynthesis (Wu-Pong, November 1994, BioPharm, 20-33). The antisenseoligonucleotide binds to target RNA by Watson Crick base-pairing andblocks gene expression by preventing ribosomal translation of the boundsequences either by steric blocking or by activating RNase H enzyme.Antisense molecules may also alter protein synthesis by interfering withRNA processing or transport from the nucleus into the cytoplasm(Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 7, 151-190).

In addition, binding of single stranded DNA to RNA may result innuclease degradation of the heteroduplex (Wu-Pong, supra; Crooke,supra). To date, the only backbone modified DNA chemistry which will actas substrates for RNase H are phosphorothioates, phosphorodithioates,and borontrifluoridates. Recently, it has been reported that 2′-arabinoand 2′-fluoro arabino-containing oligos can also activate RNase Hactivity.

A number of antisense molecules have been described that utilize novelconfigurations of chemically modified nucleotides, secondary structure,and/or RNase H substrate domains (Woolf et al., U.S. Pat. No. 5,989,912;Thompson et al., U.S. Ser. No. 60/082,404 which was filed on Apr. 20,1998; Hartmann et al., U.S. Ser. No. 60/101,174 which was filed on Sep.21, 1998) all of these are incorporated by reference herein in theirentirety.

Antisense DNA can be used to target RNA by means of DNA-RNAinteractions, thereby activating RNase H, which digests the target RNAin the duplex. Antisense DNA can be chemically synthesized or can beexpressed via the use of a single stranded DNA intracellular expressionvector or the equivalent thereof.

Triplex Forming Oligonucleotides (TFO): Single stranded oligonucleotidecan be designed to bind to genomic DNA in a sequence specific manner.TFOs can be comprised of pyrimidine-rich oligonucleotides which bind DNAhelices through Hoogsteen Base-pairing (Wu-Pong, supra). In addition,TFOs can be chemically modified to increase binding affinity to targetDNA sequences. The resulting triple helix composed of the DNA sense, DNAantisense, and TFO disrupts RNA synthesis by RNA polymerase. The TFOmechanism can result in gene expression or cell death since binding maybe irreversible (Mukhopadhyay & Roth, supra)

2′-5′ Oligoadenylates: The 2-5A system is an interferon-mediatedmechanism for RNA degradation found in higher vertebrates (Mitra et al.,1996, Proc Nat Acad Sci USA 93, 6780-6785). Two types of enzymes, 2-5Asynthetase and RNase L, are required for RNA cleavage. The 2-5Asynthetases require double stranded RNA to form 2′-5′ oligoadenylates(2-5A). 2-5A then acts as an allosteric effector for utilizing RNase L,which has the ability to cleave single stranded RNA. The ability to form2-5A structures with double stranded RNA makes this system particularlyuseful for modulation of viral replication.

(2′-5′) oligoadenylate structures can be covalently linked to antisensemolecules to form chimeric oligonucleotides capable of RNA cleavage(Torrence, supra). These molecules putatively bind and activate a2-5A-dependent RNase, the oligonucleotide/enzyme complex then binds to atarget RNA molecule which can then be cleaved by the RNase enzyme. Thecovalent attachment of 2′-5′ oligoadenylate structures is not limited toantisense applications, and can be further elaborated to includeattachment to nucleic acid molecules of the instant invention.

Enzymatic Nucleic Acid: Several varieties of naturally occurringenzymatic RNAs are presently known (Doherty and Doudna, 2001, Annu. Rev.Biophys. Biomol. Struct., 30, 457-475; Symons, 1994, Curr. Opin. Struct.Biol., 4, 322-30). In addition, several in vitro selection (evolution)strategies (Orgel, 1979, Proc. R. Soc. London, B 205, 435) have beenused to evolve new nucleic acid catalysts capable of catalyzing cleavageand ligation of phosphodiester linkages (Joyce, 1989, Gene, 82, 83-87;Beaudry et al., 1992, Science 257, 635-641; Joyce, 1992, ScientificAmerican 267, 90-97; Breaker et al., 1994, TIBTECH 12, 268; Bartel etal., 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumaret al., 1995, FASEB J, 9, 1183; Breaker, 1996, Curr. Op. Biotech., 7,442; Santoro et al., 1997, Proc. Natl. Acad. Sci., 94, 4262; Tang etal., 1997, RNA 3, 914; Nakamaye & Eckstein, 1994, supra; Long &Uhlenbeck, 1994, supra; Ishizaka et al., 1995, supra; Vaish et al.,1997, Biochemistry 36, 6495). Each can catalyze a series of reactionsincluding the hydrolysis of phosphodiester bonds in trans (and thus cancleave other RNA molecules) under physiological conditions.

The enzymatic nature of an enzymatic nucleic acid has significantadvantages, such as the concentration of nucleic acid necessary toaffect a therapeutic treatment is low. This advantage reflects theability of the enzymatic nucleic acid molecule to act enzymatically.Thus, a single enzymatic nucleic acid molecule is able to cleave manymolecules of target RNA. In addition, the enzymatic nucleic acidmolecule is a highly specific modulator, with the specificity ofmodulation depending not only on the base-pairing mechanism of bindingto the target RNA, but also on the mechanism of target RNA cleavage.Single mismatches, or base-substitutions, near the site of cleavage canbe chosen to completely eliminate catalytic activity of an enzymaticnucleic acid molecule.

Nucleic acid molecules having an endonuclease enzymatic activity areable to repeatedly cleave other separate RNA molecules in a nucleotidebase sequence-specific manner. With proper design and construction, suchenzymatic nucleic acid molecules can be targeted to any RNA transcript,and efficient cleavage achieved in vitro (Zaug et al., 324, Nature 4291986; Uhlenbeck, 1987 Nature 328, 596; Kim et al., 84 Proc. Natl. Acad.Sci. USA 8788, 1987; Dreyfus, 1988, Einstein Quart. J. Bio. Med., 6, 92;Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988;and Jefferies et al., 17 Nucleic Acids Research 1371, 1989; Chartrand etal., 1995, Nucleic Acids Research 23, 4092; Santoro et al., 1997, PNAS94, 4262).

Because of their sequence specificity, trans-cleaving enzymatic nucleicacid molecules show promise as therapeutic agents for human disease(Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294;Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037). Enzymaticnucleic acid molecule can be designed to cleave specific RNA targetswithin the background of cellular RNA. Such a cleavage event renders theRNA non-functional and abrogates protein expression from that RNA. Inthis manner, synthesis of a protein associated with a disease state canbe selectively modulated (Warashina et al., 1999, Chemistry and Biology,6, 237-250).

The present invention also features nucleic acid sensor molecules orallozymes having sensor domains comprising nucleic acid decoys and/oraptamers of the invention. Interaction of the nucleic acid sensormolecule's sensor domain with a molecular target can activate orinactivate the enzymatic nucleic acid domain of the nucleic acid sensormolecule, such that the activity of the nucleic acid sensor molecule ismodulated in the presence of the target-signaling molecule. The nucleicacid sensor molecule can be designed to be active in the presence of thetarget molecule or alternately, can be designed to be inactive in thepresence of the molecular target. For example, a nucleic acid sensormolecule is designed with a sensor domain comprising an aptamer withbinding specificity for a ligand. In a non-limiting example, interactionof the ligand with the sensor domain of the nucleic acid sensor moleculecan activate the enzymatic nucleic acid domain of the nucleic acidsensor molecule such that the sensor molecule catalyzes a reaction, forexample cleavage of RNA that encodes the ligand. In this example, thenucleic acid sensor molecule is activated in the presence of ligand, andcan be used as a therapeutic to treat a disease or condition associatedwith the ligand. Alternately, the reaction can comprise cleavage orligation of a labeled nucleic acid reporter molecule, providing a usefuldiagnostic reagent to detect the presence of ligand in a system.

RNA interference: The discussion that follows discusses the proposedmechanism of RNA interference mediated by short interfering RNA as ispresently known, and is not meant to be limiting and is not an admissionof prior art. Applicant demonstrates herein that chemically-modifiedshort interfering nucleic acids possess similar or improved capacity tomediate RNAi as do siRNA molecules and are expected to possess improvedstability and activity in vivo; therefore, this discussion is not meantto be limiting only to siRNA and can be applied to siNA as a whole. By“improved capacity to mediate RNAi” or “improved RNAi activity” is meantto include RNAi activity measured in vitro and/or in vivo where the RNAiactivity is a reflection of both the ability of the siNA to mediate RNAiand the stability of the siNAs of the invention. In this invention, theproduct of these activities can be increased in vitro and/or in vivocompared to an all RNA siRNA or a siNA containing a plurality ofribonucleotides. In some cases, the activity or stability of the siNAmolecule can be decreased (i.e., less than ten-fold), but the overallactivity of the siNA molecule is enhanced in vitro and/or in vivo.

RNA interference refers to the process of sequence specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes whichis commonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or the random integration of transposonelements into a host genome via a cellular response that specificallydestroys homologous single-stranded RNA or viral genomic RNA. Thepresence of dsRNA in cells triggers the RNAi response though a mechanismthat has yet to be fully characterized. This mechanism appears to bedifferent from the interferon response that results from dsRNA-mediatedactivation of protein kinase PKR and 2′,5′-oligoadenylate synthetaseresulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as Dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from Dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes. Dicer has also been implicated in the excision of 21- and22-nucleotide small temporal RNAs (stRNAs) from precursor RNA ofconserved structure that are implicated in translational control(Hutvagner et al., 2001, Science, 293, 834). The RNAi response alsofeatures an endonuclease complex containing a siRNA, commonly referredto as an RNA-induced silencing complex (RISC), which mediates cleavageof single-stranded RNA having sequence homologous to the siRNA. Cleavageof the target RNA takes place in the middle of the region complementaryto the guide sequence of the siRNA duplex (Elbashir et al., 2001, GenesDev., 15, 188). In addition, RNA interference can also involve small RNA(e.g., micro-RNA or miRNA) mediated gene silencing, presumably thoughcellular mechanisms that regulate chromatin structure and therebyprevent transcription of target gene sequences (see for exampleAllshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science,297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall etal., 2002, Science, 297, 2232-2237). As such, siNA molecules of theinvention can be used to mediate gene silencing via interaction with RNAtranscripts or alternately by interaction with particular genesequences, wherein such interaction results in gene silencing either atthe transcriptional level or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans. Wiannyand Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated bydsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describeRNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001,Nature, 411, 494, describe RNAi induced by introduction of duplexes ofsynthetic 21-nucleotide RNAs in cultured mammalian cells including humanembryonic kidney and HeLa cells. Recent work in Drosophila embryoniclysates has revealed certain requirements for siRNA length, structure,chemical composition, and sequence that are essential to mediateefficient RNAi activity. These studies have shown that 21 nucleotidesiRNA duplexes are most active when containing two 2-nucleotide3′-terminal nucleotide overhangs. Furthermore, substitution of one orboth siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishesRNAi activity, whereas substitution of 3′-terminal siRNA nucleotideswith deoxy nucleotides was shown to be tolerated. Mismatch sequences inthe center of the siRNA duplex were also shown to abolish RNAi activity.In addition, these studies also indicate that the position of thecleavage site in the target RNA is defined by the 5′-end of the siRNAguide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J,20, 6877). Other studies have indicated that a 5′-phosphate on thetarget-complementary strand of a siRNA duplex is required for siRNAactivity and that ATP is utilized to maintain the 5′-phosphate moiety onthe siRNA (Nykanen et al., 2001, Cell, 107, 309); however, siRNAmolecules lacking a 5′-phosphate are active when introduced exogenously,suggesting that 5′-phosphorylation of siRNA constructs may occur invivo.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs(“small” refers to nucleic acid motifs no more than 100 nucleotides inlength, preferably no more than 80 nucleotides in length, and mostpreferably no more than 50 nucleotides in length; e.g., individual siNAoligonucleotide sequences or siNA sequences synthesized in tandem) arepreferably used for exogenous delivery. The simple structure of thesemolecules increases the ability of the nucleic acid to invade targetedregions of protein and/or RNA structure. Exemplary molecules of theinstant invention are chemically synthesized, and others can similarlybe synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. All of these references areincorporated herein by reference. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoronucleotides. Table II outlines the amounts and the contact times of thereagents used in the synthesis cycle. Alternatively, syntheses at the0.2 μmol scale can be performed on a 96-well plate synthesizer, such asthe instrument produced by Protogene (Palo Alto, Calif.) with minimalmodification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol)of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyltetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycleof 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-foldexcess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-foldexcess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used ineach coupling cycle of deoxy residues relative to polymer-bound5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc.synthesizer, determined by colorimetric quantitation of the tritylfractions, are typically 97.5-99%. Other oligonucleotide synthesisreagents for the 394 Applied Biosystems, Inc. synthesizer include thefollowing: detritylation solution is 3% TCA in methylene chloride (ABI);capping is performed with 16% N-methyl imidazole in THF (ABI) and 10%acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solutionis 16.9 mM 12, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems,Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directlyfrom the reagent bottle. S-Ethyltetrazole solution (0.25 M inacetonitrile) is made up from the solid obtained from AmericanInternational Chemical, Inc. Alternately, for the introduction ofphosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows:the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mLglass screw top vial and suspended in a solution of 40% aqueousmethylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C.,the supernatant is removed from the polymer support. The support iswashed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and thesupernatant is then added to the first supernatant. The combinedsupernatants, containing the oligoribonucleotide, are dried to a whitepowder.

The method of synthesis used for RNA including certain siNA molecules ofthe invention follows the procedure as described in Usman et al., 1987,J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res.,18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of commonnucleic acid protecting and coupling groups, such as dimethoxytrityl atthe 5′-end, and phosphoramidites at the 3′-end. In a non-limitingexample, small scale syntheses are conducted on a 394 AppliedBiosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5min coupling step for alkylsilyl protected nucleotides and a 2.5 mincoupling step for 2′-O-methylated nucleotides. Table II outlines theamounts and the contact times of the reagents used in the synthesiscycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a96-well plate synthesizer, such as the instrument produced by Protogene(Palo Alto, Calif.) with minimal modification to the cycle. A 33-foldexcess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can beused in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol)of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess ofS-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in eachcoupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer include the following:detritylation solution is 3% TCA in methylene chloride (ABI); capping isperformed with 16% N-methyl imidazole in THF (ABI) and 10% aceticanhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM12, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.).Burdick & Jackson Synthesis Grade acetonitrile is used directly from thereagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) ismade up from the solid obtained from American International Chemical,Inc. Alternately, for the introduction of phosphorothioate linkages,Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M inacetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-potprotocol. For the two-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10min. After cooling to −20° C., the supernatant is removed from thepolymer support. The support is washed three times with 1.0 mL ofEtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to thefirst supernatant. The combined supernatants, containing theoligoribonucleotide, are dried to a white powder. The base deprotectedoligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mLTEA.3HF to provide a 1.4 M HF concentration) and heated to 65° C. After1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL)at 65° C. for 15 minutes. The vial is brought to room temperatureTEA.3HF (0.1 mL) is added and the vial is heated at 65° C. for 15minutes. The sample is cooled at −20° C. and then quenched with 1.5 MNH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenprewashed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5% TFA for13 minutes. The cartridge is then washed again with water, saltexchanged with 1 M NaCl and washed with water again. The oligonucleotideis then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al.,1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in theart will recognize that the scale of synthesis can be adapted to belarger or smaller than the example described above including but notlimited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention canbe synthesized separately and joined together post-synthetically, forexample, by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International PCT publication No. WO 93/23569; Shabarova et al.,1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides& Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204),or by hybridization following synthesis and/or deprotection.

The siNA molecules of the invention can also be synthesized via a tandemsynthesis methodology as described in Example 1 herein, wherein bothsiNA strands are synthesized as a single contiguous oligonucleotidefragment or strand separated by a cleavable linker which is subsequentlycleaved to provide separate siNA fragments or strands that hybridize andpermit purification of the siNA duplex. The linker can be apolynucleotide linker or a non-nucleotide linker. The tandem synthesisof siNA as described herein can be readily adapted to bothmultiwell/multiplate synthesis platforms such as 96 well or similarlylarger multi-well platforms. The tandem synthesis of siNA as describedherein can also be readily adapted to large scale synthesis platformsemploying batch reactors, synthesis columns and the like.

A siNA molecule can also be assembled from two distinct nucleic acidstrands or fragments wherein one fragment includes the sense region andthe second fragment includes the antisense region of the RNA molecule.

The nucleic acid molecules of the present invention can be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H(for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al.,1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purifiedby gel electrophoresis using general methods or can be purified by highpressure liquid chromatography (HPLC; see Wincott et al., supra, thetotality of which is hereby incorporated herein by reference) andre-suspended in water.

In another aspect of the invention, siNA molecules of the invention areexpressed from transcription units inserted into DNA or RNA vectors. Therecombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Therecombinant vectors capable of expressing the siNA molecules can bedelivered as described herein, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of siNA molecules.

Preparation of Lipid Nanoparticle (LNP) Compositions

In one embodiment, the invention features a process for producing alipid nanoparticle composition of the invention. The process typicallyincludes providing an aqueous solution comprising a biologically activemolecule of the invention (e.g., a siNA, miRNA, siRNA, or RNAiinhibitor) and/or a carrier molecule of the invention, in a firstreservoir, the first reservoir in fluid communication with an organiclipid solution in a second reservoir, and mixing the aqueous solutionwith the organic lipid solution, followed by an incubation step, adiafiltration step, and a final concentration step. In one embodiment,the aqueous solution such as a buffer, comprises a biologically activemolecule and/or carrier molecule, such that the biologically activemolecule is encapsulated in the lipid nanoparticle as a result of theprocess. In certain embodiments, the carrier molecule is dissolved inthe organic lipid solution in the second reservoir.

In another embodiment, the invention features apparatus for producing alipid nanoparticle (LNP) composition including a biologically activemolecule. The apparatus typically includes a first reservoir for holdingan aqueous solution, and a second reservoir for holding an organic lipidsolution, wherein the aqueous solution includes the biologically activemolecule. The apparatus also typically includes a pump mechanismconfigured to pump the aqueous and the organic lipid solutions into amixing region or mixing chamber at substantially equal flow rates. Inone embodiment, the mixing region or mixing chamber comprises a Tcoupling or equivalent thereof, which allows the aqueous and organicfluid streams to combine as input into the T connector and the resultingcombined aqueous and organic solutions to exit out of the T connectorinto a collection reservoir or equivalent thereof. In operation, theorganic lipid solution mixes with the aqueous solution in the mixingregion to form a desired lipid nanoparticle composition afterincubation, difiltration, and concentration.

In one embodiment, the invention features a process for synthesizing alipid nanoparticle composition of the invention comprising: (a)providing an aqueous solution comprising a biologically active moleculeof the invention (e.g., a siNA, miRNA, siRNA, or RNAi inhibitor) and/orcarrier molecule of the invention; (b) providing an organic solutioncomprising LNP components of the invention (see for example LNPcomponents shown in Table IV); (c) mixing the aqueous solution with theorganic solution; (d) incubating the resulting combined aqueous andorganic solution prior to (e) dilution; (f) ultrafiltration; and (g)final concentration under conditions suitable to produce the lipidnanoparticle composition. In one embodiment, the biologically activemolecule is encapsulated in the lipid nanoparticle as a result of theprocess.

In one embodiment, the present invention provides a method for thepreparation of a lipid nanoparticle (LNP) composition comprising abiologically active molecule, comprising: (a) preparing a solution ofthe biologically active molecule(s) and/or carrier molecule(s) ofinterest (e.g., siNA, miRNA, RNAi inhibitor) in a suitable buffer; (b)preparing a solution of lipid components (e.g. CLinDMA, DSPC,Cholesterol, PEG-DMG), and/or Linoleyl alcohol) in a suitable buffer;(c) mixing the lipid component solution and the biologically activemolecule solution together under conditions suitable for particleformation; (d) incubating the resulting mixture prior to (e) dilutionwith a suitable buffer; (f) ultrafiltration; and (g) final concentrationof the LNP composition (see for example Table VI).

In one embodiment, the buffer of (a) is an aqueous buffer such as acitrate buffer. In another embodiment, the buffer of (b) comprises anorganic alcohol such as ethanol. In one embodiment, the mixing in (c)comprises utilizing a pumping apparatus that combines a first fluidstream of the solution of (a) and a second fluid stream of the solutionof (b) into a mixing region at substantially equal flow rates to formthe lipid nanoparticle composition. In another embodiment, theincubation of (d) comprises allowing the resulting in-process solutionof (c) to stand in a vessel for about 12 to about 100 hours (preferablyabout 12 to about 24 hours) at about room temperature and optionallyprotected from light. In one embodiment, the dilution (e) involvesdilution with aqueous buffer (e.g., citrate buffer) using a pump system(such as a diaphragm pump). In one embodiment, ultrafiltration (f)comprises concentration of the diluted LNP solution followed bydiafiltration, for example using a suitable pumping system (e.g.,pumping apparatus such as a Quatroflow pump or equivalent thereof) inconjunction with a suitable ultrafiltration membrane (e.g., GE NPUFP-100-C-35A or equivalent thereof).

In another group of embodiments, the present invention provides a methodfor the preparation of a lipid nanoparticle (LNP), comprising: (a)preparing a mixture comprising cationic lipids and noncationic lipids inan organic solvent; (b) contacting an aqueous solution of molecule(s) ofinterest (e.g., biologically active molecules and/or carrier molecules)with the mixture in step (a) to provide a clear single phase; and (c)removing the organic solvent to provide a suspension of molecule-lipidparticles, wherein the molecule of interest is encapsulated in a lipidbilayer, and the particles are stable in serum and have a size of fromabout 50 to about 150 nm or alternately 50 to about 600 nm.

The selection of an organic solvent will typically involve considerationof solvent polarity and the ease with which the solvent can be removedat the later stages of particle formation. The organic solvent, which isalso used as a solubilizing agent, is in an amount sufficient to providea clear single phase mixture of biologically active molecules andlipids. Suitable solvents include, but are not limited to, chloroform,dichloromethane, diethylether, cyclohexane, cyclopentane, benzene,toluene, methanol, or other aliphatic alcohols such as propanol,isopropanol, butanol, tert-butanol, iso-butanol, pentanol and hexanol.Combinations of two or more solvents can also be used in the presentinvention.

Contacting the molecules of interest with the organic solution ofcationic and neutral lipids is accomplished by mixing together a firstsolution of the molecule of interest, which is typically an aqueoussolution, and a second organic solution of the lipids. One of skill inthe art will understand that this mixing can take place by any number ofmethods, for example by mechanical means such as by using vortex mixers.

After the molecule of interest has been contacted with the organicsolution of lipids, the organic solvent is removed, thus forming anaqueous suspension of serum-stable molecule-lipid particles. The methodsused to remove the organic solvent will typically involve evaporation atreduced pressures or blowing a stream of inert gas (e.g., nitrogen orargon) across the mixture.

The formulation or compositions thus formed will typically be sized fromabout 50 nm to 150 nm or alternately from about 50 nm to 600 nm or fromabout 5 to 1000 nm. To achieve further size reduction or homogeneity ofsize in the particles, sizing can be conducted as described above.

In other embodiments, the methods will further comprise adding nonlipidpolycations which are useful to effect the transformation of cells usingthe present compositions. Examples of suitable nonlipid polycationsinclude, but are limited to, hexadimethrine bromide (sold under thebrandname POLYBRENE®, from Aldrich Chemical Co., Milwaukee, Wis., USA)or other salts of hexadimethrine. Other suitable polycations include,for example, salts of poly-L-ornithine, poly-L-arginine, poly-L-lysine,poly-D-lysine, polyallylamine and polyethyleneimine.

In certain embodiments, the formation of the lipid nanoparticle (LNP)compositions can be carried out either in a mono-phase system (e.g., aBligh and Dyer monophase or similar mixture of aqueous and organicsolvents) or in a two-phase system with suitable mixing.

When formation of the lipid nanoparticle (LNP) is carried out in amono-phase system, the cationic lipids and molecules of interest areeach dissolved in a volume of the mono-phase mixture. Combination of thetwo solutions provides a single mixture in which the complexes form.Alternatively, the complexes can form in two-phase mixtures in which thecationic lipids bind to the molecule (which is present in the aqueousphase), and “pull” it into the organic phase.

In another embodiment, the present invention provides a method for thepreparation of lipid nanoparticle (LNP) compositions, comprising: (a)contacting molecules of interest (e.g., biologically active moleculesand/or carrier molecules) with a solution comprising noncationic lipidsand a detergent to form a molecule-lipid mixture; (b) contactingcationic lipids with the molecule-lipid mixture to neutralize a portionof the negative charge of the molecules of interest and form acharge-neutralized mixture of molecules and lipids; and (c) removing thedetergent from the charge-neutralized mixture to provide the lipidnanoparticle (LNP) composition.

In one group of embodiments, the solution of neutral lipids anddetergent is an aqueous solution. Contacting the molecules of interest(e.g., biologically active molecules and/or carrier molecules) with thesolution of neutral lipids and detergent is typically accomplished bymixing together a first solution of the molecule of interest and asecond solution of the lipids and detergent. One of skill in the artwill understand that this mixing can take place by any number ofmethods, for example, by mechanical means such as by using vortexmixers. Preferably, the molecule solution is also a detergent solution.The amount of neutral lipid which is used in the present method istypically determined based on the amount of cationic lipid used, and istypically of from about 0.2 to 5 times the amount of cationic lipid,preferably from about 0.5 to about 2 times the amount of cationic lipidused.

The molecule-lipid mixture thus formed is contacted with cationic lipidsto neutralize a portion of the negative charge which is associated withthe molecule of interest (e.g., biologically active molecules and/orcarrier molecules or other polyanionic materials) present. The amount ofcationic lipids used is typically the amount sufficient to neutralize atleast 50% of the negative charge of the molecule of interest.Preferably, the negative charge will be at least 70% neutralized, morepreferably at least 90% neutralized. Cationic lipids which are useful inthe present invention include, for example, compounds having any offormulae CLI-CLXXIX, DODAC, DOTMA, DDAB, DOTAP, DC-Chol, DMOBA, CLinDMA,and DMRIE. These lipids and related analogs have been described in U.S.Ser. No. 08/316,399; U.S. Pat. Nos. 5,208,036, 5,264,618, 5,279,833 and5,283,185, the disclosures of which are incorporated by reference intheir entireties herein. Additionally, a number of commercialpreparations of cationic lipids are available and can be used in thepresent invention. These include, for example, LIPOFECTIN®(commerciallyavailable cationic liposomes comprising DOTMA and DOPE, from GIBCO/BRL,Grand Island, N.Y., USA); LIPOFECTAMINE® (commercially availablecationic liposomes comprising DOSPA and DOPE, from GIBCO/BRL); andTRANSFECTAM® (commercially available cationic lipids comprising DOGS inethanol from Promega Corp., Madison, Wis., USA).

Contacting the cationic lipids with the molecule-lipid mixture can beaccomplished by any of a number of techniques, preferably by mixingtogether a solution of the cationic lipid and a solution containing themolecule-lipid mixture. Upon mixing the two solutions (or contacting inany other manner), a portion of the negative charge associated with themolecule of interest is neutralized.

After the cationic lipids have been contacted with the molecule-lipidmixture, the detergent (or combination of detergent and organic solvent)is removed, thus forming the formulation or composition. The methodsused to remove the detergent typically involve dialysis. When organicsolvents are present, removal is typically accomplished by evaporationat reduced pressures or by blowing a stream of inert gas (e.g., nitrogenor argon) across the mixture.

The lipid nanoparticle (LNP) composition particles thus formed aretypically sized from about 50 nm to several microns. To achieve furthersize reduction or homogeneity of size in the particles, the lipidnanoparticle (LNP) composition particles can be sonicated, filtered orsubjected to other sizing techniques which are used in liposomalformulations and are known to those of skill in the art.

In other embodiments, the methods further comprise adding nonlipidpolycations which are useful to affect the lipofection of cells usingthe present compositions. Examples of suitable nonlipid polycationsinclude, hexadimethrine bromide (sold under the brandname POLYBRENE®,from Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts ofhexadimethrine. Other suitable polycations include, for example, saltsof poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine,polyallylamine and polyethyleneimine. Addition of these salts ispreferably after the particles have been formed.

In another aspect, the present invention provides methods for thepreparation of formulated siNA compositions, comprising: (a) contactingan amount of cationic lipids with siNA in a solution; the solutioncomprising from about 15-35% water and about 65-85% organic solvent andthe amount of cationic lipids being sufficient to produce a +/− chargeratio of from about 0.85 to about 2.0, to provide a hydrophobiclipid-siNA complex; (b) contacting the hydrophobic, lipid-siNA complexin solution with neutral lipids, to provide a siNA-lipid mixture; and(c) removing the organic solvents from the lipid-siNA mixture to provideformulated siNA composition particles.

In another aspect, the present invention provides methods for thepreparation of formulated siNA/carrier compositions, comprising: (a)contacting an amount of cationic lipids with siNA/carrier in a solution;the solution comprising from about 15-35% water and about 65-85% organicsolvent and the amount of cationic lipids being sufficient to produce a+/− charge ratio of from about 0.85 to about 2.0, to provide ahydrophobic lipid-siNA/carrier complex; (b) contacting the hydrophobic,lipid-siNA/carrier complex in solution with neutral lipids, to provide asiNA/carrier-lipid mixture; and (c) removing the organic solvents fromthe lipid-siNA/carrier mixture to provide formulated siNA/carriercomposition particles.

In another aspect, the present invention provides methods for thepreparation of formulated carrier molecule compositions, comprising: (a)contacting an amount of cationic lipids with carrier molecule(s) in asolution; the solution comprising from about 15-35% water and about65-85% organic solvent and the amount of cationic lipids beingsufficient to produce a +/− charge ratio of from about 0.85 to about2.0, to provide a hydrophobic lipid-carrier complex; (b) contacting thehydrophobic, lipid-carrier complex in solution with neutral lipids, toprovide a siNA-carrier mixture; and (c) removing the organic solventsfrom the lipid-carrier mixture to provide formulated carrier compositionparticles.

The siNA, carrier molecules, neutral lipids, cationic lipids and organicsolvents which are useful in this aspect of the invention are the sameas those described for the methods above which used detergents. In onegroup of embodiments, the solution of step (a) is a mono-phase. Inanother group of embodiments, the solution of step (a) is two-phase.

In one embodiment, the cationic lipids used in a formulation of theinvention are selected from a compound having Formula CLI, CLII, CLIII,CLIV, CLV, CLVI, CLVII, CLVIII, CLIX, CLX, CLXI, CLXII, CLXIII, CLXIV,CLXV, CLXVI, CLXVII, CLXVIII, CLXIX, CLXX, CLXXI, CLXXII, CLXXIII,CLXXIV, CLXXV, CLXXVI, CLXXVII, CLXXVIII, CLXXIX, CLXXX, CLXXXI,CLXXXII, CLXXXIII, CLXXXIV, CLXXXV, CLXXXVI, CLXXXVII, CLXXXVIII,CLXXXIX, CLXXXX, CLXXXXI, CLXXXXII and DODAC, DDAB, DOTMA, DODAP,DOCDAP, DLINDAP, DOSPA, DMRIE, DOGS, DMOBA, CLinDMA, and combinationsthereof. In one embodiment, the noncationic lipids are selected fromESM, DOPE, DOPC, DSPC, polyethylene glycol-based polymers (e.g., PEG2000, PEG 5000 or PEG-modified diacylglycerols),distearoylphosphatidylcholine (DSPC), cholesterol, and combinationsthereof. In one embodiment, the organic solvents are selected frommethanol, chloroform, methylene chloride, ethanol, diethyl ether andcombinations thereof.

In one embodiment, the cationic lipid is a compound having Formula CLI,CLII, CLIII, CLIV, CLV, CLVI, CLVII, CLVIII, CLIX, CLX, CLXI, CLXII,CLXIII, CLXIV, CLXV, CLXVI, CLXVII, CLXVIII, CLXVII, CLXVIII, CLXIX,CLXX, CLXXI, CLXXII, CLXXIII, CLXXIV, CLXXV, CLXXVI, CLXXVII, CLXXVIII,CLXXIX, CLXXX, CLXXXI, CLXXXII, CLXXXIII, CLXXXIV, CLXXXV, CLXXXVI,CLXXXVII, CLXXXVIII, CLXXXIX or DODAC, DOTAP, DODAP, DOCDAP, DLINDAP,DDAB, DOTMA, DOSPA, DMRIE, DOGS or combinations thereof; the noncationiclipid is ESM, DOPE, DAG-PEGs, distearoylphosphatidylcholine (DSPC),cholesterol, or combinations thereof (e.g. DSPC and DAG-PEGs); and theorganic solvent is methanol, chloroform, methylene chloride, ethanol,diethyl ether or combinations thereof.

As above, contacting the siNA and/or carrier with the cationic lipids istypically accomplished by mixing together a first solution of siNAand/or carrier and a second solution of the lipids, preferably bymechanical means such as by using vortex mixers. The resulting mixturecontains complexes as described above. These complexes are thenconverted to particles by the addition of neutral lipids and the removalof the organic solvent. The addition of the neutral lipids is typicallyaccomplished by simply adding a solution of the neutral lipids to themixture containing the complexes. A reverse addition can also be used.Subsequent removal of organic solvents can be accomplished by methodsknown to those of skill in the art and also described above.

The amount of neutral lipids which is used in this aspect of theinvention is typically an amount of from about 0.2 to about 15 times theamount (on a mole basis) of cationic lipids which was used to providethe charge-neutralized lipid-nucleic acid complex. Preferably, theamount is from about 0.5 to about 9 times the amount of cationic lipidsused.

In yet another aspect, the present invention provides formulated siNAand/or carrier compositions which are prepared by the methods describedabove. In these embodiments, the formulated siNA and/or carriercompositions are either net charge neutral or carry an overall chargewhich provides the formulated siNA and/or carrier compositions withgreater lipofection activity. In one embodiment, the noncationic lipidis egg sphingomyelin and the cationic lipid is DODAC. In one embodiment,the noncationic lipid is a mixture of DSPC and cholesterol, and thecationic lipid is DOTMA. In another embodiment, the noncationic lipidcan further comprise cholesterol.

Non-limiting examples of methods of preparing nucleic acid formulationsare disclosed in U.S. Pat. No. 5,976,567, U.S. Pat. No. 5,981,501 andPCT Patent Publication No. WO 96/40964, the teachings of all of whichare incorporated in their entireties herein by reference. Cationiclipids that are useful in the present invention can be any of a numberof lipid species which carry a net positive charge at a selected pH,such as physiological pH. Suitable cationic lipids include, but are notlimited to, a compound having any of Formulae CLI-CLXXXXVI, DODAC,DOTMA, DDAB, DOTAP, DODAP, DOCDAP, DLINDAP, DOSPA, DOGS, DC-Chol andDMRIE, as well as other cationic lipids described herein, orcombinations thereof. A number of these cationic lipids and relatedanalogs, which are also useful in the present invention, have beendescribed in U.S. Ser. No. 08/316,399; U.S. Pat. Nos. 5,208,036,5,264,618, 5,279,833 and 5,283,185, the disclosures of which areincorporated herein by reference. Additionally, a number of commercialpreparations of cationic lipids are available and can be used in thepresent invention. These include, for example, LIPOFECTIN® (commerciallyavailable cationic liposomes comprising DOTMA and DOPE, from GIBCO/BRL,Grand Island, N.Y., USA); LIPOFECTAMINE® (commercially availablecationic liposomes comprising DOSPA and DOPE, from GIBCO/BRL); andTRANSFECTAM® (commercially available cationic liposomes comprising DOGSfrom Promega Corp., Madison, Wis., USA).

The noncationic lipids used in the present invention can be any of avariety of neutral uncharged, zwitterionic or anionic lipids capable ofproducing a stable complex. They are preferably neutral, although theycan alternatively be positively or negatively charged. Examples ofnoncationic lipids useful in the present invention includephospholipid-related materials, such as lecithin,phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin,cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylet-hanolamine (POPE) anddioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal). Noncationiclipids or sterols such as cholesterol may be present. Additionalnonphosphorous containing lipids are, e.g., stearylamine, dodecylamine,hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecylstereate, isopropyl myristate, amphoteric acrylic polymers,triethanolamine-lauryl sulfate, alkylaryl sulfate polyethyloxylatedfatty acid amides, dioctadecyldimethyl ammonium bromide and the like,diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, and cerebrosides. Other lipids such aslysophosphatidylcholine and lysophosphatidylethanolamine may be present.Noncationic lipids also include polyethylene glycol-based polymers suchas PEG 2000, PEG 5000 and polyethylene glycol conjugated tophospholipids or to ceramides (referred to as PEG-Cer), as described inco-pending U.S. Ser. No. 08/316,429, incorporated herein by reference.

In one embodiment, the noncationic lipids are diacylphosphatidylcholine(e.g., distearoylphosphatidylcholine, dioleoylphosphatidylcholine,dipalmitoylphosphatidylcholine or dilinoleoylphosphatidylcholine),diacylphosphatidylethanolamine (e.g., dioleoylphosphatidylethanolamineand palmitoyloleoylphosphatidylethanolamine), ceramide or sphingomyelin.The acyl groups in these lipids are preferably acyl groups derived fromfatty acids having about C10 to about C24 carbon chains. In oneembodiment, the acyl groups are lauroyl, myristoyl, palmitoyl, stearoylor oleoyl. In additional embodiments, the noncationic lipid comprisescholesterol, 1,2-sn-dioleoylphosphatidylethanol-amine, or eggsphingomyelin (ESM).

In addition to cationic and neutral lipids, the formulation orcomposition s of the present invention comprise a polyethyleneglycol(PEG) conjugate. The PEG conjugate can comprise adiacylglycerol-polyethyleneglycol conjugate, i.e., a DAG-PEG conjugate.The term “diacylglycerol” refers to a compound having 2-fatty acylchains, R1 and R2, both of which have independently between 2 and 30carbons bonded to the 1- and 2-position of glycerol by ester linkages.The acyl groups can be saturated or have varying degrees ofunsaturation. Diacylglycerols have the following general formula VIII:

wherein R1 and R2 are each an alkyl, substituted alkyl, aryl,substituted aryl, lipid, or a ligand. In one embodiment, R1 and R2 areeach independently a C2 to C30 alkyl group.

In one embodiment, the DAG-PEG conjugate is a dilaurylglycerol (C12)-PEGconjugate, a dimyristylglycerol (C14)-PEG conjugate, adipalmitoylglycerol (C16)-PEG conjugate, a disterylglycerol (C18)-PEGconjugate, a PEG-dilaurylglycamide conjugate (C12), aPEG-dimyristylglycamide conjugate (C14), a PEG-dipalmitoylglycamideconjugate (C16), or a PEG-disterylglycamide (C18). Those of skill in theart will readily appreciate that other diacylglycerols can be used inthe DAG-PEG conjugates of the present invention.

The PEG conjugate can alternatively comprise a conjugate other than aDAG-PEG conjugate, such as a PEG-cholesterol conjugate or a PEG-DMBconjugate.

In addition to the foregoing components, the formulation or compositions or LNPs of the present invention can further comprise cationicpoly(ethylene glycol) (PEG) lipids, or CPLs, that have been designed forinsertion into lipid bilayers to impart a positive charge (see forexample Chen, et al., 2000, Bioconj. Chem. 11, 433-437). Suitableformulations for use in the present invention, and methods of making andusing such formulations are disclosed, for example in U.S. applicationSer. No. 09/553,639, which was filed Apr. 20, 2000, and PCT PatentApplication No. CA 00/00451, which was filed Apr. 20, 2000 and whichpublished as WO 00/62813 on Oct. 26, 2000, the teachings of each ofwhich is incorporated herein in its entirety by reference.

The formulation or composition s of the present invention, i.e., thoseformulation or composition s or LNPs containing DAG-PEG conjugates, canbe made using any of a number of different methods. For example, thelipid-nucleic acid particles can be produced via hydrophobic siNA-lipidintermediate complexes. The complexes are preferably charge-neutralized.Manipulation of these complexes in either detergent-based or organicsolvent-based systems can lead to particle formation in which thenucleic acid is protected.

The present invention provides a method of preparing serum-stableformulation or composition s (or lipid nanoparticles, LNPs), includingformulations that undergo pH-dependent phase transition, in which thebiologically active molecule is encapsulated in a lipid bilayer and isprotected from degradation. Additionally, the formulated particlesformed in the present invention are preferably neutral ornegatively-charged at physiological pH. For in vivo applications,neutral particles are advantageous, while for in vitro applications theparticles are more preferably negatively charged. This provides thefurther advantage of reduced aggregation over the positively-chargedliposome formulations in which a biologically active molecule can beencapsulated in cationic lipids.

The formulated particles and LNPs made by the methods of this inventionhave a size of about 50 to about 600 nm or more, with certain of theparticles being about 65 to 85 nm. The particles can be formed by eithera detergent dialysis method or by a modification of a reverse-phasemethod which utilizes organic solvents to provide a single phase duringmixing of the components. Without intending to be bound by anyparticular mechanism of formation, a biologically active molecule iscontacted with a detergent solution of cationic lipids to form a coatedmolecular complex. These coated molecules can aggregate and precipitate.However, the presence of a detergent reduces this aggregation and allowsthe coated molecules to react with excess lipids (typically, noncationiclipids) to form particles in which the biologically active molecule isencapsulated in a lipid bilayer. The methods described below for theformation of formulation or composition s using organic solvents followa similar scheme.

In some embodiments, the particles are formed using detergent dialysis.Thus, the present invention provides a method for the preparation ofserum-stable formulation or composition s (including formulations thatundergo pH-dependent phase transition) comprising: (a) combining amolecule of interest with cationic lipids in a detergent solution toform a coated molecule-lipid complex; (b) contacting noncationic lipidswith the coated molecule-lipid complex to form a detergent solutioncomprising a molecule-lipid complex and noncationic lipids; and (c)dialyzing the detergent solution of step (b) to provide a solution ofserum-stable molecule-lipid particles, wherein the molecule of interestis encapsulated in a lipid bilayer and the particles have a size of fromabout 50 to about 600 nm. In one embodiment, the particles have a sizeof from about 50 to about 150 nm.

An initial solution of coated molecule-lipid complexes is formed, forexample, by combining the molecule of interest with the cationic lipidsin a detergent solution.

In these embodiments, the detergent solution is preferably an aqueoussolution of a neutral detergent having a critical micelle concentrationof 15-300 mM, more preferably 20-50 mM. Examples of suitable detergentsinclude, for example,N,N′-((octanoylimino)-bis-(trimethylene))-bis-(D-gluconamide) (BIGCHAP);BRIJ 35; Deoxy-BIGCHAP; dodecylpoly(ethylene glycol) ether; Tween 20;Tween 40; Tween 60; Tween 80; Tween 85; Mega 8; Mega 9; Zwittergent®3-08; Zwittergent® 3-10; Triton X-405; hexyl-, heptyl-, octyl- andnonyl-beta-D-glucopyranoside; and heptylthioglucopyranoside. In oneembodiment, the detergent is octyl β-D-glucopyranoside or Tween-20. Theconcentration of detergent in the detergent solution is typically about100 mM to about 2 M, preferably from about 200 mM to about 1.5 M.

The cationic lipids and molecules to be encapsulated will typically becombined to produce a charge ratio (+/−) of about 1:1 to about 20:1,preferably in a ratio of about 1:1 to about 12:1, and more preferably ina ratio of about 2:1 to about 6:1. Additionally, the overallconcentration of the molecules of interest in solution will typically befrom about 25 μg/mL to about 1 mg/mL, preferably from about 25 μg/mL toabout 500 μg/mL, and more preferably from about 100 μg/mL to about 250μg/mL. The combination of molecules and cationic lipids in detergentsolution is kept, typically at room temperature, for a period of timewhich is sufficient for the coated complexes to form. Alternatively, themolecules and cationic lipids can be combined in the detergent solutionand warmed to temperatures of up to about 37° C. For molecules which areparticularly sensitive to temperature, the coated complexes can beformed at lower temperatures, typically down to about 4° C.

In one embodiment, the molecule to lipid ratios (mass/mass ratios) in aformed formulation or composition will range from about 0.01 to about0.08. The ratio of the starting materials also falls within this rangebecause the purification step typically removes the unencapsulatedmolecule as well as the empty liposomes. In another embodiment, theformulation or composition preparation uses about 400 μg siNA per 10 mgtotal lipid or a molecule to lipid ratio of about 0.01 to about 0.08and, more preferably, about 0.04, which corresponds to 1.25 mg of totallipid per 50 μg of siNA.

The detergent solution of the coated molecule-lipid complexes is thencontacted with neutral lipids to provide a detergent solution ofmolecule-lipid complexes and neutral lipids. The neutral lipids whichare useful in this step include, among others,diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cardiolipin, and cerebrosides. In preferredembodiments, the neutral lipids are diacylphosphatidylcholine,diacylphosphatidylethanolamine, ceramide or sphingomyelin. The acylgroups in these lipids are preferably acyl groups derived from fattyacids having C10-C24 carbon chains. More preferably the acyl groups arelauroyl, myristoyl, palmitoyl, stearoyl or oleoyl. In preferredembodiments, the neutral lipid is1,2-sn-dioleoylphosphatidylethanolamine (DOPE), palmitoyl oleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC),distearoylphosphatidylcholine (DSPC), cholesterol, or a mixture thereof.In the most preferred embodiments, the siNA-lipid particles arefusogenic particles with enhanced properties in vivo and the neutrallipid is DSPC or DOPE. As explained above, the siNA-lipid particles ofthe present invention can further comprise PEG conjugates, such asDAG-PEG conjugates, PEG-cholesterol conjugates, and PEG-DMB conjugates.In addition, the siNA-lipid particles of the present invention canfurther comprise cholesterol.

The amount of neutral lipid which is used in the present methods istypically about 0.5 to about 10 mg of total lipids to 50 μg of themolecule of interest. Preferably the amount of total lipid is from about1 to about 5 mg per 50 μg of the molecule of interest.

Following formation of the detergent solution of molecule-lipidcomplexes and neutral lipids, the detergent is removed, preferably bydialysis. The removal of the detergent results in the formation of alipid-bilayer which surrounds the molecule of interest providingserum-stable molecule-lipid particles which have a size of from about 50nm to about 150 or 50 nm to about 600 nm. The particles thus formed donot aggregate and are optionally sized to achieve a uniform particlesize.

The serum-stable molecule-lipid particles can be sized by any of themethods available for sizing liposomes as are known in the art. Thesizing can be conducted in order to achieve a desired size range andrelatively narrow distribution of particle sizes.

Several techniques are available for sizing the particles to a desiredsize. One sizing method, used for liposomes and equally applicable tothe present particles is described in U.S. Pat. No. 4,737,323,incorporated herein by reference. Sonicating a particle suspensioneither by bath or probe sonication produces a progressive size reductiondown to particles of less than about 50 nm in size. Homogenization isanother method which relies on shearing energy to fragment largerparticles into smaller ones. In a typical homogenization procedure,particles are recirculated through a standard emulsion homogenizer untilselected particle sizes, typically between about 60 and 80 nm, areobserved. In both methods, the particle size distribution can bemonitored by conventional laser-beam particle size discrimination, orQELS.

When the “size” of a particle is described herein, reference is beingmade to the mean size of a distribution of particles, for example asmeasured by conventional laser-beam particle size discrimination, orQELS. Thus, a particle having a size of 50 nm to 150 nm can refer to acollection of particles of that type having a mean size of 50 nm to 150nm, for example as measured by conventional laser-beam particle sizediscrimination, or QELS. Preferably the standard deviation of thedistribution is less than 50%, more preferably less than 30%, and evenmore preferably less than 10% of the mean size of the distribution.

Extrusion of the particles through a small-pore polycarbonate membraneor an asymmetric ceramic membrane is also an effective method forreducing particle sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired particle size distribution is achieved. Theparticles can be extruded through successively smaller-pore membranes,to achieve a gradual reduction in size.

A variety of general methods for making formulated siNA composition-CPLs(CPL-containing formulated siNA compositions) are discussed herein. Twogeneral techniques include “post-insertion” technique, that is,insertion of a CPL into for example, a preformed formulated siNAcomposition, and the “standard” technique, wherein the CPL is includedin the lipid mixture during for example, the formulated siNA compositionformation steps. The post-insertion technique results in formulated siNAcompositions having CPLs mainly in the external face of the formulatedsiNA composition bilayer membrane, whereas standard techniques provideformulated siNA compositions having CPLs on both internal and externalfaces.

In particular, “post-insertion” involves forming formulated siNAcompositions (by any method), and incubating the pre-formed formulatedsiNA compositions in the presence of CPL under appropriate conditions(preferably 2-3 hours at 60° C.). Between 60-80% of the CPL can beinserted into the external leaflet of the recipient vesicle, givingfinal concentrations up to about 5 to 10 mol % (relative to totallipid). The method is especially useful for vesicles made fromphospholipids (which can contain cholesterol) and also for vesiclescontaining PEG-lipids (such as PEG-DAGs).

In an example of a “standard” technique, the CPL-formulated siNAcompositions of the present invention can be formed by extrusion. Inthis embodiment, all of the lipids including the CPL, are co-dissolvedin chloroform, which is then removed under nitrogen followed by highvacuum. The lipid mixture is hydrated in an appropriate buffer, andextruded through two polycarbonate filters with a pore size of 100 nm.The resulting formulated siNA compositions contain CPL on both of theinternal and external faces. In yet another standard technique, theformation of CPL-formulated siNA compositions can be accomplished usinga detergent dialysis or ethanol dialysis method, for example, asdiscussed in U.S. Pat. Nos. 5,976,567 and 5,981,501, both of which areincorporated by reference in their entireties herein.

The formulated siNA compositions of the present invention can beadministered either alone or in mixture with aphysiologically-acceptable carrier (such as physiological saline orphosphate buffer) selected in accordance with the route ofadministration and standard pharmaceutical practice. Generally, normalsaline will be employed as the pharmaceutically acceptable carrier.Other suitable carriers include, e.g., water, buffered water, 0.4%saline, 0.3% glycine, and the like, including glycoproteins for enhancedstability, such as albumin, lipoprotein, globulin, etc.

The pharmaceutical carrier is generally added following formulated siNAcomposition formation. Thus, after the formulated siNA composition isformed, the formulated siNA composition can be diluted intopharmaceutically acceptable carriers such as normal saline.

The concentration of formulated siNA compositions in the pharmaceuticalformulations can vary widely, i.e., from less than about 0.05%, usuallyat or at least about 2-5% to as much as 10 to 30% by weight and will beselected primarily by fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected. For example, theconcentration can be increased to lower the fluid load associated withtreatment. This may be particularly desirable in patients havingatherosclerosis-associated congestive heart failure or severehypertension. Alternatively, formulated siNA compositions composed ofirritating lipids can be diluted to low concentrations to lesseninflammation at the site of administration.

As described above, the formulated siNA compositions of the presentinvention comprise DAG-PEG conjugates. It is often desirable to includeother components that act in a manner similar to the DAG-PEG conjugatesand that serve to prevent particle aggregation and to provide a meansfor increasing circulation lifetime and increasing the delivery of theformulated siNA compositions to the target tissues. Such componentsinclude, but are not limited to, PEG-lipid conjugates, such asPEG-ceramides or PEG-phospholipids (such as PEG-PE), gangliosideGM1-modified lipids or ATTA-lipids to the particles. Typically, theconcentration of the component in the particle will be about 1-20% and,more preferably from about 3-10%.

The pharmaceutical compositions of the present invention can besterilized by conventional, well known sterilization techniques. Aqueoussolutions can be packaged for use or filtered under aseptic conditionsand lyophilized, the lyophilized preparation being combined with asterile aqueous solution prior to administration. The compositions cancontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, andcalcium chloride. Additionally, the particle suspension can includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as alphatocopherol and water-soluble iron-specificchelators, such as ferrioxamine, are suitable

In another example of their use, formulation or composition s can beincorporated into a broad range of topical dosage forms including, butnot limited to, gels, oils, emulsions and the like. For instance, thesuspension containing the formulation or composition s can be formulatedand administered as topical creams, pastes, ointments, gels, lotions andthe like.

Once formed, the formulation or composition s of the present inventionare useful for the introduction of biologically active molecules intocells. Accordingly, the present invention also provides methods forintroducing a biologically active molecule into a cell. The methods arecarried out in vitro or in vivo by first forming the formulation orcomposition s as described above and then contacting the formulation orcomposition s with the cells for a period of time sufficient fortransfection to occur.

The formulation or composition s of the present invention can beadsorbed to almost any cell type with which they are mixed or contacted.Once adsorbed, the formulations can either be endocytosed by a portionof the cells, exchange lipids with cell membranes, or fuse with thecells. Transfer or incorporation of the biologically active moleculeportion of the formulation can take place via any one of these pathways.In particular, when fusion takes place, the particle membrane isintegrated into the cell membrane and the contents of the particle,i.e., biologically active molecules, combine with the intracellularfluid, for example, the cytoplasm. The serum stable formulation orcomposition s that undergo pH-dependent phase transition demonstrate anincrease in cell fusion at early endosomal pH (i.e., about pH 5.5-6.5),resulting in efficient delivery of the contents of the particle, i.e.,biologically active molecules, to the cell.

Using the Endosomal Release Parameter (ERP) assay of the presentinvention, the transfection efficiency of the formulation or compositionor other lipid-based carrier system can be optimized. More particularly,the purpose of the ERP assay is to distinguish the effect of variouscationic lipids and helper lipid components of formulation orcomposition s based on their relative effect on binding/uptake or fusionwith/destabilization of the endosomal membrane. This assay allows one todetermine quantitatively how each component of the formulation orcomposition or other lipid-based carrier system effects transfectionefficacy, thereby optimizing the formulation or composition s or otherlipid-based carrier systems. As explained herein, the Endosomal ReleaseParameter or, alternatively, ERP is defined as: Reporter GeneExpression/Cell divided by formulation or composition Uptake/Cell.

It will be readily apparent to those of skill in the art that anyreporter gene (e.g., luciferase, beta-galactosidase, green fluorescentprotein, etc.) can be used in the assay. In addition, the lipidcomponent (or, alternatively, any component of the formulation orcomposition) can be labeled with any detectable label provided the doesinhibit or interfere with uptake into the cell. Using the ERP assay ofthe present invention, one of skill in the art can assess the impact ofthe various lipid components (e.g., cationic lipid, neutral lipid,PEG-lipid derivative, PEG-DAG conjugate, ATTA-lipid derivative, calcium,CPLs, cholesterol, etc.) on cell uptake and transfection efficiencies,thereby optimizing the formulated siNA composition. By comparing theERPs for each of the various formulation or composition s, one canreadily determine the optimized system, e.g., the formulation orcomposition that has the greatest uptake in the cell coupled with thegreatest transfection efficiency.

Suitable labels for carrying out the ERP assay of the present inventioninclude, but are not limited to, spectral labels, such as fluorescentdyes (e.g., fluorescein and derivatives, such as fluoresceinisothiocyanate (FITC) and Oregon Green9; rhodamine and derivatives, suchTexas red, tetrarhodimine isothiocynate (TRITC), etc., digoxigenin,biotin, phycoerythrin, AMCA, CyDyes, and the like; radiolabels, such as³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, etc.; enzymes, such as horse radishperoxidase, alkaline phosphatase, etc.; spectral colorimetric labels,such as colloidal gold or colored glass or plastic beads, such aspolystyrene, polypropylene, latex, etc. The label can be coupleddirectly or indirectly to a component of the formulation or compositionusing methods well known in the art. As indicated above, a wide varietyof labels can be used, with the choice of label depending on sensitivityrequired, ease of conjugation with the formulated siNA composition,stability requirements, and available instrumentation and disposalprovisions.

In addition, the transfection efficiency of the formulation orcomposition or other lipid-based carrier system can be determined bymeasuring the stability of the composition in serm and/or measuring thepH dependent phase transition of the formulation or composition, whereina determination that the formulation or composition is stable in serumand a determination that the formulation or composition undergoes aphase transition at about pH 5.5-6.5 indicates that the formulation orcomposition will have increased transfection efficiency. The serumstability of the formulation or composition can be measured using, forexample, an assay that measures the relative turbidity of thecomposition in serum and determining that the turbidity of thecomposition in serum remains constant over time. The pH dependent phasetransition of the formulation or composition can be measured using anassay that measures the relative turbidity of the composition atdifferent pH over time and determining that the turbidity changes whenthe pH differs from physiologic pH.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules (e.g., siNA, miRNA, RNAiinhibitor, antisense, aptamer, decoy, ribozyme, 2-5A, triplex formingoligonucleotide, or other nucleic acid molecule) with modifications(base, sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency (see e.g., Eckstein etal., International Publication No. WO 92/07065; Perrault et al., 1990Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman andCedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al.,supra; all of which are incorporated by reference herein). All of theabove references describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules described herein. Modifications that enhance their efficacy incells, and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for areview see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994,Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35,14090). Sugar modification of nucleic acid molecules have beenextensively described in the art (see Eckstein et al., InternationalPublication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344,565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren,Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. InternationalPublication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 andBeigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al.,International PCT publication No. WO 97/26270; Beigelman et al., U.S.Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al.,International PCT Publication No. WO 98/13526; Thompson et al., U.S.Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al.,1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers(Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev.Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5,1999-2010; all of the references are hereby incorporated in theirtotality by reference herein). Such publications describe generalmethods and strategies to determine the location of incorporation ofsugar, base and/or phosphate modifications and the like into nucleicacid molecules without modulating catalysis, and are incorporated byreference herein. In view of such teachings, similar modifications canbe used as described herein to modify the siNA nucleic acid molecules ofthe instant invention so long as the ability of siNA to promote RNAicells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonatelinkages improves stability, excessive modifications can cause sometoxicity or decreased activity. Therefore, when designing nucleic acidmolecules, the amount of these internucleotide linkages should beminimized. The reduction in the concentration of these linkages shouldlower toxicity, resulting in increased efficacy and higher specificityof these molecules.

Polynucleotides (e.g., siNA, miRNA, RNAi inhibitor, antisense, aptamer,decoy, ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleicacid molecule) having chemical modifications that maintain or enhanceactivity are provided. Such a nucleic acid is also generally moreresistant to nucleases than an unmodified nucleic acid. Accordingly, thein vitro and/or in vivo activity should not be significantly lowered. Incases in which modulation is the goal, therapeutic nucleic acidmolecules delivered exogenously should optimally be stable within cellsuntil translation of the target RNA has been modulated long enough toreduce the levels of the undesirable protein. This period of time variesbetween hours to days depending upon the disease state. Improvements inthe chemical synthesis of RNA and DNA (Wincott et al., 1995, NucleicAcids Res. 23, 2677; Caruthers et al., 1992, Methods in Enzymology 211,3-19 (incorporated by reference herein)) have expanded the ability tomodify nucleic acid molecules by introducing nucleotide modifications toenhance their nuclease stability, as described above.

In one embodiment, nucleic acid molecules of the invention include oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clampnucleotides. A G-clamp nucleotide is a modified cytosine analog whereinthe modifications confer the ability to hydrogen bond both Watson-Crickand Hoogsteen faces of a complementary guanine within a duplex, see forexample Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. Asingle G-clamp analog substitution within an oligonucleotide can resultin substantially enhanced helical thermal stability and mismatchdiscrimination when hybridized to complementary oligonucleotides. Theinclusion of such nucleotides in nucleic acid molecules of the inventionresults in both enhanced affinity and specificity to nucleic acidtargets, complementary sequences, or template strands. In anotherembodiment, nucleic acid molecules of the invention include one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleicacid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (seefor example Wengel et al., International PCT Publication No. WO 00/66604and WO 99/14226).

In another embodiment, the invention features conjugates and/orcomplexes of siNA molecules of the invention. Such conjugates and/orcomplexes can be used to facilitate delivery of siNA molecules into abiological system, such as a cell. The conjugates and complexes providedby the instant invention can impart therapeutic activity by transferringtherapeutic compounds across cellular membranes, altering thepharmacokinetics, and/or modulating the localization of nucleic acidmolecules of the invention. The present invention encompasses the designand synthesis of novel conjugates and complexes for the delivery ofmolecules, including, but not limited to, small molecules, lipids,cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids,antibodies, toxins, negatively charged polymers and other polymers, forexample proteins, peptides, hormones, carbohydrates, polyethyleneglycols, or polyamines, across cellular membranes. In general, thetransporters described are designed to be used either individually or aspart of a multi-component system, with or without degradable linkers.These compounds are expected to improve delivery and/or localization ofnucleic acid molecules of the invention into a number of cell typesoriginating from different tissues, in the presence or absence of serum(see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of themolecules described herein can be attached to biologically activemolecules via linkers that are biodegradable, such as biodegradablenucleic acid linker molecules.

The term “biodegradable linker” as used herein, refers to a nucleic acidor non-nucleic acid linker molecule that is designed as a biodegradablelinker to connect one molecule to another molecule, for example, abiologically active molecule to a siNA molecule of the invention or thesense and antisense strands of a siNA molecule of the invention. Thebiodegradable linker is designed such that its stability can bemodulated for a particular purpose, such as delivery to a particulartissue or cell type. The stability of a nucleic acid-based biodegradablelinker molecule can be modulated by using various chemistries, forexample combinations of ribonucleotides, deoxyribonucleotides, andchemically-modified nucleotides, such as 2′-O-methyl, 2′-fluoro,2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified orbase modified nucleotides. The biodegradable nucleic acid linkermolecule can be a dimer, trimer, tetramer or longer nucleic acidmolecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length,or can comprise a single nucleotide with a phosphorus-based linkage, forexample, a phosphoramidate or phosphodiester linkage. The biodegradablenucleic acid linker molecule can also comprise nucleic acid backbone,nucleic acid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in abiological system, for example, enzymatic degradation or chemicaldegradation.

The term “phospholipid” as used herein, refers to a hydrophobic moleculecomprising at least one phosphorus group. For example, a phospholipidcan comprise a phosphorus-containing group and saturated or unsaturatedalkyl group, optionally substituted with OH, COOH, oxo, amine, orsubstituted or unsubstituted aryl groups.

Therapeutic nucleic acid molecules (e.g., siNA, miRNA, RNAi inhibitor,antisense, aptamer, decoy, ribozyme, 2-5A, triplex formingoligonucleotide, or other nucleic acid molecule) delivered exogenouslyoptimally are stable within cells until reverse transcription of the RNAhas been modulated long enough to reduce the levels of the RNAtranscript. The nucleic acid molecules are resistant to nucleases inorder to function as effective intracellular therapeutic agents.Improvements in the chemical synthesis of nucleic acid moleculesdescribed in the instant invention and in the art have expanded theability to modify nucleic acid molecules by introducing nucleotidemodifications to enhance their nuclease stability as described above.

In yet another embodiment, siNA molecules having chemical modificationsthat maintain or enhance enzymatic activity of proteins involved in RNAiare provided. Such nucleic acids are also generally more resistant tonucleases than unmodified nucleic acids. Thus, in vitro and/or in vivothe activity should not be significantly lowered.

Use of the nucleic acid-based molecules of the invention will lead tobetter treatments by affording the possibility of combination therapies(e.g., multiple siNA molecules targeted to different genes; nucleic acidmolecules coupled with known small molecule modulators; or intermittenttreatment with combinations of molecules, including different motifsand/or other chemical or biological molecules).

In another aspect a polynucleotide molecule of the invention (e.g.,siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,triplex forming oligonucleotide, or other nucleic acid molecule)comprises one or more 5′ and/or a 3′-cap structure, for example, on onlythe sense siNA strand, the antisense siNA strand, or both siNA strands.

By “cap structure” is meant chemical modifications, which have beenincorporated at either terminus of the oligonucleotide (see, forexample, Adamic et al., U.S. Pat. No. 5,998,203, incorporated byreference herein). These terminal modifications protect the nucleic acidmolecule from exonuclease degradation, and may help in delivery and/orlocalization within a cell. The cap may be present at the 5′-terminus(5′-cap) or at the 3′-terminal (3′-cap) or may be present on bothtermini. In non-limiting examples, the 5′-cap includes, but is notlimited to, glyceryl, inverted deoxy abasic residue (moiety);4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide,4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitolnucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide;phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety;3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety;3′-2′-inverted abasic moiety; 1,4-butanediol phosphate;3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate;3′-phosphorothioate; phosphorodithioate; or bridging or non-bridgingmethylphosphonate moiety

Non-limiting examples of the 3′-cap include, but are not limited to,glyceryl, inverted deoxy abasic residue (moiety), 4′,5′-methylenenucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide,carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propylphosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate;1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitolnucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

By the term “non-nucleotide” is meant any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound is abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine and therefore lacks a base at the 1′-position.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, includingstraight-chain, branched-chain, and cyclic alkyl groups. Preferably, andunless expressly stated to to the contrary, the alkyl group has 1 to 12carbons. More preferably, it is a lower alkyl of from 1 to 7 carbons,more preferably 1 to 4 carbons. The alkyl group can be substituted orunsubstituted. When substituted the substituted group(s) is preferably,hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino, or SH. The termalso includes alkenyl groups that are unsaturated hydrocarbon groupscontaining at least one carbon-carbon double bond, includingstraight-chain, branched-chain, and cyclic groups. Preferably, thealkenyl group has 1 to 12 carbons. More preferably, it is a loweralkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. Thealkenyl group may be substituted or unsubstituted. When substituted thesubstituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S,NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includesalkynyl groups that have an unsaturated hydrocarbon group containing atleast one carbon-carbon triple bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkynyl group has 1to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7carbons, more preferably 1 to 4 carbons. The alkynyl group may besubstituted or unsubstituted. When substituted the substituted group(s)is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino orSH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. An “aryl” group refers to anaromatic group that has at least one ring having a conjugated pielectron system and includes carbocyclic aryl, heterocyclic aryl andbiaryl groups, all of which may be optionally substituted. The preferredsubstituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH,OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An“alkylaryl” group refers to an alkyl group (as described above)covalently joined to an aryl group (as described above). Carbocyclicaryl groups are groups wherein the ring atoms on the aromatic ring areall carbon atoms. The carbon atoms are optionally substituted.Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms asring atoms in the aromatic ring and the remainder of the ring atoms arecarbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen,and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo,pyrimidyl, pyrazinyl, imidazolyl and the like, all optionallysubstituted. An “amide” refers to an —C(O)—NH—R, where R is eitheralkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR,where R is either alkyl, aryl, alkylaryl or hydrogen.

By “nucleotide” as used herein is as recognized in the art to includenatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1′ position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see, for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22,2183. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, inosine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others(Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents.

In one embodiment, the invention features modified polynucleotidemolecules (e.g., siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleic acidmolecule), with phosphate backbone modifications comprising one or morephosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkylsilyl, substitutions. For a review ofoligonucleotide backbone modifications, see Hunziker and Leumann, 1995,Nucleic Acid Analogues: Synthesis and Properties, in Modern SyntheticMethods, VCH, 331-417, and Mesmaeker et al., 1994, Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39.

By “abasic” is meant sugar moieties lacking a base or having otherchemical groups in place of a base at the 1′ position, see for exampleAdamic et al., U.S. Pat. No. 5,998,203.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine,guanine, thymine, or uracil joined to the 1′ carbon ofβ-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains amodification in the chemical structure of an unmodified nucleotide base,sugar and/or phosphate. Non-limiting examples of modified nucleotidesare shown by Formulae I-VII and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the presentinvention, by “amino” is meant 2′—NH₂ or 2′-O—NH₂, which can be modifiedor unmodified. Such modified groups are described, for example, inEckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S.Pat. No. 6,248,878, which are both incorporated by reference in theirentireties.

Various modifications to nucleic acid siNA structure can be made toenhance the utility of these molecules. Such modifications will enhanceshelf-life, half-life in vitro, stability, and ease of introduction ofsuch oligonucleotides to the target site, e.g., to enhance penetrationof cellular membranes, and confer the ability to recognize and bind totargeted cells.

By “cholesterol derivative” is meant, any compound consistingessentially of a cholesterol structure, including additions,substitutions and/or deletions thereof. The term cholesterol derivativeherein also includes steroid hormones and bile acids as are generallyrecognized in the art.Administration of Formulated siNA Compositions

A formulation or composition of the invention can be adapted for use toprevent, inhibit, or reduce any trait, disease or condition that isrelated to or will respond to the levels of target gene expression in acell or tissue, alone or in combination with other therapies.

In one embodiment, formulation or compositions can be administered tocells by a variety of methods known to those of skill in the art,including, but not restricted to, by injection, by iontophoresis or byincorporation into other vehicles, such as biodegradable polymers,hydrogels, cyclodextrins (see for example Gonzalez et al., 1999,Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCTpublication Nos. WO 03/47518 and WO 03/46185). In one embodiment, aformulation or composition s of the invention are complexed withmembrane disruptive agents such as those described in U.S. PatentApplication Publication No. 20010007666, incorporated by referenceherein in its entirety including the drawings. In another embodiment,the membrane disruptive agent or agents and the biologically activemolecule are also complexed with a cationic lipid or helper lipidmolecule, such as those lipids described in U.S. Pat. No. 6,235,310,incorporated by reference herein in its entirety including the drawings.

In one embodiment, delivery systems of the invention include, forexample, aqueous and nonaqueous gels, creams, multiple emulsions,microemulsions, ointments, aqueous and nonaqueous solutions, lotions,aerosols, hydrocarbon bases and powders, and can contain excipients suchas solubilizers, permeation enhancers (e.g., fatty acids, fatty acidesters, fatty alcohols and amino acids), and hydrophilic polymers (e.g.,polycarbophil and polyvinylpyrolidone). In one embodiment, thepharmaceutically acceptable carrier is a transdermal enhancer.

In one embodiment, delivery systems of the invention include patches,tablets, suppositories, pessaries, gels and creams, and can containexcipients such as solubilizers and enhancers (e.g., propylene glycol,bile salts and amino acids), and other vehicles (e.g., polyethyleneglycol, fatty acid esters and derivatives, and hydrophilic polymers suchas hydroxypropylmethylcellulose and hyaluronic acid).

In one embodiment, the invention features a pharmaceutical compositioncomprising one or more formulated siNA compositions of the invention inan acceptable carrier, such as a stabilizer, buffer, and the like. Theformulation or composition s of the invention can be administered andintroduced to a subject by any standard means, with or withoutstabilizers, buffers, and the like, to form a pharmaceuticalcomposition. The compositions of the present invention can also beformulated and used as creams, gels, sprays, oils and other suitablecompositions for topical, dermal, or transdermal administration as isknown in the art.

In one embodiment, the invention also includes pharmaceuticallyacceptable formulations of the compounds described. These formulationsinclude salts of the above compounds, e.g., acid addition salts, forexample, salts of hydrochloric, hydrobromic, acetic acid, and benzenesulfonic acid.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemic orlocal administration, into a cell or subject, including for example ahuman. Suitable forms, in part, depend upon the use or the route ofentry, for example oral, transdermal, or by injection. Such forms shouldnot prevent the composition or formulation from reaching a target cell(i.e., a cell to which the siNA is desirable for delivery). For example,pharmacological compositions injected into the blood stream should besoluble. Other factors are known in the art, and include considerationssuch as toxicity and forms that prevent the composition or formulationfrom exerting its effect.

In one embodiment, formulation or composition s of the invention areadministered to a subject by systemic administration in apharmaceutically acceptable composition or formulation. By “systemicadministration” is meant in vivo systemic absorption or accumulation ofdrugs in the blood stream followed by distribution throughout the entirebody. Administration routes that lead to systemic absorption include,without limitation: intravenous, subcutaneous, intraperitoneal,inhalation, oral, intrapulmonary and intramuscular. Each of theseadministration routes exposes the siNA molecules of the invention to anaccessible diseased tissue. The rate of entry of a drug into thecirculation has been shown to be a function of molecular weight or size.

By “pharmaceutically acceptable formulation” or “pharmaceuticallyacceptable composition” is meant, a composition or formulation thatallows for the effective distribution of the formulated molecular Acompositions of the instant invention in the physical location mostsuitable for their desired activity. Non-limiting examples of agentssuitable for formulation with the formulation or composition s of theinstant invention include: P-glycoprotein inhibitors (such as PluronicP85); biodegradable polymers, such as poly(DL-lactide-coglycolide)microspheres for sustained release delivery (Emerich, D F et al, 1999,Cell Transplant, 8, 47-58); and loaded nanoparticles, such as those madeof polybutylcyanoacrylate. Other non-limiting examples of deliverystrategies for the nucleic acid molecules of the instant inventioninclude material described in Boado et al., 1998, J. Pharm. Sci., 87,1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge etal., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug DeliveryRev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26,4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

The present invention also includes compositions prepared for storage oradministration that include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985),hereby incorporated by reference herein. For example, preservatives,stabilizers, dyes and flavoring agents can be provided. These includesodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Inaddition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors that those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the formulated siNA composition.

The formulation or composition s of the invention can be administeredorally, topically, parenterally, by inhalation or spray, or rectally indosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants and/or vehicles. Theterm parenteral as used herein includes percutaneous, subcutaneous,intravascular (e.g., intravenous), intramuscular, or intrathecalinjection or infusion techniques and the like. In addition, there isprovided a pharmaceutical formulation comprising a formulation orcomposition of the invention and a pharmaceutically acceptable carrier.One or more formulation or composition s of the invention can be presentin association with one or more non-toxic pharmaceutically acceptablecarriers and/or diluents and/or adjuvants, and if desired other activeingredients. The pharmaceutical compositions containing formulation orcomposition s of the invention can be in a form suitable for oral use,for example, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The formulation or composition s of the invention can also beadministered in the form of suppositories, e.g., for rectaladministration of the drug. These compositions can be prepared by mixingthe drug with a suitable non-irritating excipient that is solid atordinary temperatures but liquid at the rectal temperature and willtherefore melt in the rectum to release the drug. Such materials includecocoa butter and polyethylene glycols.

Formulation or composition s of the invention can be administeredparenterally in a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per subject perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient.

It is understood that the specific dose level for any particular subjectdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

The formulation or composition s of the present invention can also beadministered to a subject in combination with other therapeuticcompounds to increase the overall therapeutic effect. The use ofmultiple compounds to treat an indication can increase the beneficialeffects while reducing the presence of side effects.

EXAMPLES Example 1 Identification of Potential siNA Target Sites in anyRNA Sequence

The sequence of an RNA target of interest, such as a viral or human mRNAtranscript (e.g., any of sequences referred to herein by GenBankAccession Number), is screened for target sites, for example by using acomputer folding algorithm. In a non-limiting example, the sequence of agene or RNA gene transcript derived from a database, such as Genbank, isused to generate siNA targets having complementarity to the target. Suchsequences can be obtained from a database, or can be determinedexperimentally as known in the art. Target sites that are known, forexample, those target sites determined to be effective target sitesbased on studies with other nucleic acid molecules, for exampleribozymes or antisense, or those targets known to be associated with adisease, trait, or condition such as those sites containing mutations ordeletions, can be used to design siNA molecules targeting those sites.Various parameters can be used to determine which sites are the mostsuitable target sites within the target RNA sequence. These parametersinclude but are not limited to secondary or tertiary RNA structure, thenucleotide base composition of the target sequence, the degree ofhomology between various regions of the target sequence, or the relativeposition of the target sequence within the RNA transcript. Based onthese determinations, any number of target sites within the RNAtranscript can be chosen to screen siNA molecules for efficacy, forexample by using in vitro RNA cleavage assays, cell culture, or animalmodels. In a non-limiting example, anywhere from 1 to 1000 target sitesare chosen within the transcript based on the size of the siNA constructto be used. High throughput screening assays can be developed forscreening siNA molecules using methods known in the art, such as withmulti-well or multi-plate assays to determine efficient reduction intarget gene expression.

Example 2 Selection of siNA Molecule Target Sites in a RNA

The following non-limiting steps can be used to carry out the selectionof siNAs targeting a given gene sequence or transcript.

1. The target sequence is parsed in silico into a list of all fragmentsor subsequences of a particular length, for example 23 nucleotidefragments, contained within the target sequence. This step is typicallycarried out using a custom Perl script, but commercial sequence analysisprograms such as Oligo, MacVector, or the GCG Wisconsin Package can beemployed as well.2. In some instances the siNAs correspond to more than one targetsequence; such would be the case for example in targeting differenttranscripts of the same gene, targeting different transcripts of morethan one gene, or for targeting both the human gene and an animalhomolog. In this case, a subsequence list of a particular length isgenerated for each of the targets, and then the lists are compared tofind matching sequences in each list. The subsequences are then rankedaccording to the number of target sequences that contain the givensubsequence; the goal is to find subsequences that are present in mostor all of the target sequences. Alternately, the ranking can identifysubsequences that are unique to a target sequence, such as a mutanttarget sequence. Such an approach would enable the use of siNA to targetspecifically the mutant sequence and not effect the expression of thenormal sequence.3. In some instances the siNA subsequences are absent in one or moresequences while present in the desired target sequence; such would bethe case if the siNA targets a gene with a paralogous family member thatis to remain untargeted. As in case 2 above, a subsequence list of aparticular length is generated for each of the targets, and then thelists are compared to find sequences that are present in the target genebut are absent in the untargeted paralog.4. The ranked siNA subsequences can be further analyzed and rankedaccording to GC content. A preference can be given to sites containing30-70% GC, with a further preference to sites containing 40-60% GC.5. The ranked siNA subsequences can be further analyzed and rankedaccording to self-folding and internal hairpins. Weaker internal foldsare preferred; strong hairpin structures are to be avoided.6. The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have runs of GGG or CCC in the sequence. GGG(or even more Gs) in either strand can make oligonucleotide synthesisproblematic and can potentially interfere with RNAi activity, so it isavoided whenever better sequences are available. CCC is searched in thetarget strand because that will place GGG in the antisense strand.7. The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have the dinucleotide UU (uridinedinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end ofthe sequence (to yield 3′ UU on the antisense sequence). These sequencesallow one to design siNA molecules with terminal TT thymidinedinucleotides.8. Four or five target sites are chosen from the ranked list ofsubsequences as described above. For example, in subsequences having 23nucleotides, the right 21 nucleotides of each chosen 23-mer subsequenceare then designed and synthesized for the upper (sense) strand of thesiNA duplex, while the reverse complement of the left 21 nucleotides ofeach chosen 23-mer subsequence are then designed and synthesized for thelower (antisense) strand of the siNA duplex. If terminal TT residues aredesired for the sequence (as described in paragraph 7), then the two 3′terminal nucleotides of both the sense and antisense strands arereplaced by TT prior to synthesizing the oligos.9. The siNA molecules are screened in an in vitro, cell culture oranimal model system to identify the most active siNA molecule or themost preferred target site within the target RNA sequence.10. Other design considerations can be used when selecting targetnucleic acid sequences, see, for example, Reynolds et al., 2004, NatureBiotechnology Advanced Online Publication, 1 Feb. 2004,doi:10.1038/nbt936 and Ui-Tei et al., 2004, Nucleic Acids Research, 32,doi: 10.1093/nar/gkh247.

In an alternate approach, a pool of siNA constructs specific to a targetsequence is used to screen for target sites in cells expressing targetRNA, such as cultured Jurkat, HeLa, A549 or 293T cells. Cells expressingthe target RNA are transfected with the pool of siNA constructs andcells that demonstrate a phenotype associated with target inhibition aresorted. The pool of siNA constructs can be expressed from transcriptioncassettes inserted into appropriate vectors. The siNA from cellsdemonstrating a positive phenotypic change (e.g., decreasedproliferation, decreased target mRNA levels or decreased target proteinexpression), are sequenced to determine the most suitable target site(s)within the target RNA sequence.

In one embodiment, siNA molecules of the invention are selected usingthe following methodology. The following guidelines were compiled topredict hyper-active siNAs that contain chemical modifications describedherein. These rules emerged from a comparative analysis of hyper-active(>75% knockdown of target mRNA levels) and inactive (<75% knockdown oftarget mRNA levels) siNAs against several different targets. A total of242 siNA sequences were analyzed. Thirty-five siNAs out of 242 siNAswere grouped into hyper-active and the remaining siNAs were grouped intoinactive groups. The hyper-active siNAs clearly showed a preference forcertain bases at particular nucleotide positions within the siNAsequence. For example, A or U nucleobase was overwhelmingly present atposition 19 of the sense strand in hyper-active siNAs and opposite wastrue for inactive siNAs. There was also a pattern of a A/U rich (3 outof 5 bases as A or U) region between positions 15-19 and G/C rich regionbetween positions 1-5 (3 out of 5 bases as G or C) of the sense strandin hyperactive siNAs. As shown in Table V, 12 such patterns wereidentified that were characteristics of hyperactive siNAs. It is to benoted that not every pattern was present in each hyper-active siNA.Thus, to design an algorithm for predicting hyper-active siNAs, adifferent score was assigned for each pattern. Depending on howfrequently such patterns occur in hyper-active siNAs versus inactivesiNAs, the design parameters were assigned a score with the highestbeing 10. If a certain nucleobase is not preferred at a position, then anegative score was assigned. For example, at positions 9 and 13 of thesense strand, a G nucleotide was not preferred in hyperactive siNAs andtherefore they were given score of −3(minus 3). The differential scorefor each pattern is given in Table V. The pattern # 4 was given amaximum score of −100. This is mainly to weed out any sequence thatcontains string of 4Gs or 4Cs as they can be highly incompatible forsynthesis and can allow sequences to self-aggregate, thus rendering thesiNA inactive. Using this algorithm, the highest score possible for anysiNA is 66. As there are numerous siNA sequences possible against anygiven target of reasonable size (˜1000 nucleotides), this algorithm isuseful to generate hyper-active siNAs.

In one embodiment, rules 1-11 shown in Table V are used to generateactive siNA molecules of the invention. In another embodiment, rules1-12 shown in Table V are used to generate active siNA molecules of theinvention.

Example 3 siNA Design

siNA target sites were chosen by analyzing sequences of the target RNAsequences using the parameters described in Example 3 above andoptionally prioritizing the target sites on the basis of the rulespresented in Example 3 above, and alternately on the basis of folding(structure of any given sequence analyzed to determine siNAaccessibility to the target), or by using a library of siNA molecules asdescribed in Example 3, or alternately by using an in vitro siNA systemas described in Example 6 herein. siNA molecules were designed thatcould bind each target and are selected using the algorithm above andare optionally individually analyzed by computer folding to assesswhether the siNA molecule can interact with the target sequence.Chemical modification criteria were applied in designing chemicallymodified siNA molecules based on stabilization chemistry motifsdescribed herein (see for example Table I). Varying the length of thesiNA molecules can be chosen to optimize activity. Generally, asufficient number of complementary nucleotide bases are chosen to bindto, or otherwise interact with, the target RNA, but the degree ofcomplementarity can be modulated to accommodate siNA duplexes or varyinglength or base composition. By using such methodologies, siNA moleculescan be designed to target sites within any known RNA sequence, forexample those RNA sequences corresponding to the any gene transcript.

Target RNA sequences were analysed to generate targets from which doublestranded siNA and multifunctional molecules were designed. To generatesynthetic siNA constructs, the algorithm described in Example 3 wasutilized to pick active double stranded constructs and chemicallymodified versions thereof. Multifunctional siNAs were designed bysearching for homologous sites between different target sequences (e.g.,from about 5 to about 15 nucleotide regions of shared homology betweentargets) and allowing for non-canonical base pairs (e.g. up to 3 wobblebase pairing (G:U) or mismatched base pairs (e.g. up to 2 mismatches).

Chemically modified siNA constructs were designed as described herein toprovide nuclease stability for systemic administration in vivo and/orimproved pharmacokinetic, localization, and delivery properties whilepreserving the ability to mediate RNAi activity. Chemical modificationsas described herein are introduced synthetically using synthetic methodsdescribed herein and those generally known in the art. The syntheticsiNA constructs are then assayed for nuclease stability in serum and/orcellular/tissue extracts (e.g. liver extracts). The synthetic siNAconstructs are also tested in parallel for RNAi activity using anappropriate assay, such as a luciferase reporter assay as describedherein or another suitable assay that can quantity RNAi activity.Synthetic siNA constructs that possess both nuclease stability and RNAiactivity can be further modified and re-evaluated in stability andactivity assays. The chemical modifications of the stabilized activesiNA constructs can then be applied to any siNA sequence targeting anychosen RNA and used, for example, in target screening assays to picklead siNA compounds for therapeutic development.

Example 4 Chemical Synthesis and Purification of siNA

siNA molecules can be designed to interact with various sites in the RNAmessage, for example, target sequences within the RNA sequencesdescribed herein. The sequence of one strand of the siNA molecule(s) iscomplementary to the target site sequences described above. The siNAmolecules can be chemically synthesized using methods described herein.Inactive siNA molecules that are used as control sequences can besynthesized by scrambling the sequence of the siNA molecules such thatit is not complementary to the target sequence. Generally, siNAconstructs can by synthesized using solid phase oligonucleotidesynthesis methods as described herein (see for example Usman et al.,U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098;6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos.6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein intheir entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in astepwise fashion using the phosphoramidite chemistry as is known in theart. Standard phosphoramidite chemistry involves the use of nucleosidescomprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl,3′-O-2-Cyanoethyl N,N-diisopropylphos-phoroamidite groups, and exocyclicamine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine,and N2-isobutyryl guanosine). Alternately, 2′-O—Silyl Ethers can be usedin conjunction with acid-labile 2′-O-orthoester protecting groups in thesynthesis of RNA as described by Scaringe supra. Differing 2′chemistries can require different protecting groups, for example2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection asdescribed by Usman et al., U.S. Pat. No. 5,631,360, incorporated byreference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′-to 5′-direction) to the solid support-bound oligonucleotide. The firstnucleoside at the 3′-end of the chain is covalently attached to a solidsupport (e.g., controlled pore glass or polystyrene) using variouslinkers. The nucleotide precursor, a ribonucleoside phosphoramidite, andactivator are combined resulting in the coupling of the secondnucleoside phosphoramidite onto the 5′-end of the first nucleoside. Thesupport is then washed and any unreacted 5′-hydroxyl groups are cappedwith a capping reagent such as acetic anhydride to yield inactive5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized toa more stable phosphate linkage. At the end of the nucleotide additioncycle, the 5′-O-protecting group is cleaved under suitable conditions(e.g., acidic conditions for trityl-based groups and fluoride forsilyl-based groups). The cycle is repeated for each subsequentnucleotide.

Modification of synthesis conditions can be used to optimize couplingefficiency, for example by using differing coupling times, differingreagent/phosphoramidite concentrations, differing contact times,differing solid supports and solid support linker chemistries dependingon the particular chemical composition of the siNA to be synthesized.Deprotection and purification of the siNA can be performed as isgenerally described in Usman et al., U.S. Pat. No. 5,831,071, U.S. Pat.No. 6,353,098, U.S. Pat. No. 6,437,117, and Bellon et al., U.S. Pat. No.6,054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No. 6,303,773, or Scaringesupra, incorporated by reference herein in their entireties.Additionally, deprotection conditions can be modified to provide thebest possible yield and purity of siNA constructs. For example,applicant has observed that oligonucleotides comprising2′-deoxy-2′-fluoro nucleotides can degrade under inappropriatedeprotection conditions. Such oligonucleotides are deprotected usingaqueous methylamine at about 35° C. for 30 minutes. If the2′-deoxy-2′-fluoro containing oligonucleotide also comprisesribonucleotides, after deprotection with aqueous methylamine at about35° C. for 30 minutes, TEA-HF is added and the reaction maintained atabout 65° C. for an additional 15 minutes. The deprotected singlestrands of siNA are purified by anion exchange to achieve a high puritywhile maintaining high yields. To form the siNA duplex molecule thesingle strands are combined in equal molar ratios in a saline solutionto form the duplex. The duplex siNA is concentrated and desalted bytangential filtration prior to lyophilization.

Manufacture of siNA Compositions

In a non-limiting example, for each siNA composition, the twoindividual, complementary strands of the siNA are synthesized separatelyusing solid phase synthesis, then purified separately by ion exchangechromatography. The complementary strands are annealed to form thedouble strand (duplex). The duplex is then ultrafiltered and lyophilizedto form the solid siNA composition (e.g., pharmaceutical composition). Anon-limiting example of the manufacturing process is shown in the flowdiagram in Table VI.

Solid Phase Synthesis

The single strand oligonucleotides are synthesized using phosphoramiditechemistry on an automated solid-phase synthesizer, such as an AmershamPharmacia AKTA Oligopilot (e.g., Oligopilot or Oligopilot 100 plus). Anadjustable synthesis column is packed with solid support derivatizedwith the first nucleoside residue. Synthesis is initiated bydetritylation of the acid labile 5′-O-dimethoxytrityl group to releasethe 5′-hydroxyl. Phosphoramidite and a suitable activator inacetonitrile are delivered simultaneously to the synthesis columnresulting in coupling of the amidite to the 5′-hydroxyl. The column isthen washed with acetonitrile. Iodine is pumped through the column tooxidize the phosphite triester linkage P(III) to its phosphotriesterP(V) analog. Unreacted 5′-hydroxyl groups are capped using reagents suchas acetic anhydride in the presence of 2,6-lutidine andN-methylimidazole. The elongation cycle resumes with the detritylationstep for the next phosphoramidite incorporation. This process isrepeated until the desired sequence has been synthesized. The synthesisconcludes with the removal of the terminal dimethoxytrityl group.

Cleavage and Deprotection

On completion of the synthesis, the solid-support and associatedoligonucleotide are transferred to a filter funnel, dried under vacuum,and transferred to a reaction vessel. Aqueous base is added and themixture is heated to effect cleavage of the succinyl linkage, removal ofthe cyanoethyl phosphate protecting group, and deprotection of theexocyclic amine protection.

The following process is performed on single strands that do not containribonucleotides: After treating the solid support with the aqueous base,the mixture is filtered under vacuum to separate the solid support fromthe deprotected crude synthesis material. The solid support is thenrinsed with water which is combined with the filtrate. The resultantbasic solution is neutralized with acid to provide a solution of thecrude single strand.

The following process is performed on single strands that containribonucleotides: After treating the solid support with the aqueous base,the mixture is filtered under vacuum to separate the solid support fromthe deprotected crude synthesis material. The solid support is thenrinsed with dimethylsulfoxide (DMSO) which is combined with thefiltrate. The mixture is cooled, fluoride reagent such as triethylaminetrihydrofluoride is added, and the solution is heated. The reaction isquenched with suitable buffer to provide a solution of crude singlestrand.

Anion Exchange Purification

The solution of each crude single strand is purified usingchromatographic purification. The product is eluted using a suitablebuffer gradient. Fractions are collected in closed sanitized containers,analyzed by HPLC, and the appropriate fractions are combined to providea pool of product which is analyzed for purity (HPLC), identity (HPLC),and concentration (UV A260).

Annealing

Based on the analysis of the pools of product, equal molar amounts(calculated using the theoretical extinction coefficient) of the senseand antisense oligonucleotide strands are transferred to a reactionvessel. The solution is mixed and analyzed for purity of duplex bychromatographic methods. If the analysis indicates an excess of eitherstrand, then additional non-excess strand is titrated until duplexing iscomplete. When analysis indicates that the target product purity hasbeen achieved, the material is transferred to the tangential flowfiltration (TFF) system for concentration and desalting.

Ultrafiltration

The annealed product solution is concentrated using a TFF systemcontaining an appropriate molecular weight cut-off membrane. Followingconcentration, the product solution is desalted via diafiltration usingWFI quality water until the conductivity of the filtrate is that ofwater.

Lyophilization

The concentrated solution is transferred to sanitized trays in a shelflyophilizer. The product is then freeze-dried to a powder. The trays areremoved from the lyophilizer and transferred to a class 100 Laminar AirFlow (LAF) hood for packaging.

Packaging Drug Substance

The lyophilizer trays containing the freeze-dried product are opened ina class 100 LAF hood. The product is transferred to sanitized containersof appropriate size, which are then sealed and labeled.

Drug Substance Container Closure System

Lyophilized drug substance is bulk packaged in sanitized Nalgenecontainers with sanitized caps. The bottle size used is dependent uponthe quantity of material to be placed within it. After filling, eachbottle is additionally sealed at the closure with polyethylene tape.

Analytical Methods and Specifications Raw Material and In-ProcessMethods

Raw materials are tested for identity prior to introduction into thedrug substance manufacturing process. Critical raw materials, thoseincorporated into the drug substance molecule, are tested additionallyusing a purity test or an assay test as appropriate. In-process samplesare tested at key control points in the manufacturing process to monitorand assure the quality of the final drug substance.

Drug Substance Analytical Methods and Specifications

Controls incorporating analytical methods and acceptance criteria foroligonucleotides are established prior to clinical testing of bulk siNAcompositions. The following test methods and acceptance criteria reflectexamples of these controls. Table VII summarizes examples of materialspecifications for siNA pharmaceutical compositions.

Summary of Analytical Methods Identification (ID) Tests

ID Oligonucleotide Main Peak: The identity of the drug substance isestablished using a chromatographic method. The data used for thisdetermination is generated by one of the HPLC test methods (see PurityTests). The peak retention times of the drug substance sample and thestandard injections are compared. Drug substance identity is supportedby a favorable comparison of the main peak retention times.

Molecular Weight The identity of the drug substance is established usinga spectroscopic method. A sample of drug substance is prepared foranalysis by precipitation with aqueous ammonium acetate. The molecularweight of the drug substance is determined by mass spectrometry. Thetest is controlled to within a set number of atomic mass units from thetheoretical molecular weight.

Melting Temperature This method supports the identity of the drugsubstance by measurement of the melting temperature (Tm) of the doublestranded drug substance. A sample in solution is heated while monitoringthe ultraviolet (UV) absorbance of the solution. The Tm is marked by theinflection point of the absorbance curve as the absorbance increases dueto the dissociation of the duplex into single strands.

Assay Tests

Oligonucleotide Content: This assay determines the total oligonucleotidecontent in the drug substance. The oligonucleotide absorbs UV light witha local maximum at 260 nm. The oligonucleotide species present consistof the double stranded siRNA product and other minor relatedoligonucleotide substances from the manufacturing process, includingresidual single strands. A sample of the drug substance is accuratelyweighed, dissolved, and diluted volumetrically in water. The absorbanceis measured in a quartz cell using a UV spectrophotometer. The totaloligonucleotide assay value is calculated using the experimentallydetermined molar absorptivity of the working standard and reported inmicrograms of sodium oligonucleotide per milligram of solid drugsubstance.

Purity Tests Purity will be measured using one or more chromatographicmethods. Depending on the separation and the number of nucleic acidanalogs of the drug substance present, orthogonal separation methods maybe employed to monitor purity of the API. Separation may be achieved bythe following means:

SAX-HPLC: an ion exchange interaction between the oligonucleotidephosphodiesters and a strong anion exchange HPLC column using a bufferedsalt gradient to perform the separation.

RP-HPLC: a partitioning interaction between the oligonucleotide and ahydrophobic reversed-phase HPLC column using an aqueous buffer versusorganic solvent gradient to perform the separation.

Capillary Gel Electrophoresis (CGE): an electrophoretic separation bymolecular sieving in a buffer solution within a gel filled capillary.Separation occurs as an electrical field is applied, causing anionicoligonucleotides to separate by molecular size as they migrate throughthe gel matrix. In all separation methods, peaks elute generally inorder of oligonucleotide length and are detected by UV at 260 nm.

Other Tests

Physical Appearance The drug substance sample is visually examined. Thistest determines that the material has the character of a lyophilizedsolid, identifies the color of the solid, and determines whether anyvisible contaminants are present.

Bacterial Endotoxins Test: Bacterial endotoxin testing is performed bythe Limulus Amebocyte Lysate (LAL) assay using the kinetic turbidimetricmethod in a 96-well plate. Endotoxin limits for the drug substance willbe set appropriately such that when combined with the excipients, dailyallowable limits for endotoxin in the administered drug product are notexceeded.

Aerobic Bioburden Aerobic bioburden is performed by a contractlaboratory using a method based on USP chapter <61>.

Acetonitrile content: Residual acetonitrile analysis is performed by acontract laboratory using gas chromatography (GC). Acetonitrile is themajor organic solvent used in the upstream synthesis step althoughseveral other organic reagents are employed in synthesis. Subsequentpurification process steps typically remove solvents in the drugsubstances. Other solvents may be monitored depending on the outcome ofprocess development work. Solvents will be limited within ICH limits.

Water content: Water content is determined by volumetric Karl Fischer(KF) titration using a solid evaporator unit (oven). Water is typicallypresent in nucleic acid drug substances as several percent of thecomposition by weight, and therefore, will be monitored.

pH: The pH of reconstituted drug substance will be monitored to ensuresuitability for human injection.

Ion Content Testing for sodium, chloride, and phosphate will beperformed by a contract laboratory using standard atomic absorption andion chromatographic methods. General monitoring of ions will beperformed to ensure that the osmolality of the drug productincorporating the drug substances will be within an acceptablephysiological range.

Metals Content Testing for pertinent metals is performed by a contractlaboratory using a standard method of analysis, Inductively CoupledPlasma (ICP) spectroscopy.

Example 5 RNAi In Vitro Assay to Assess siNA Activity

An in vitro assay that recapitulates RNAi in a cell-free system is usedto evaluate siNA constructs targeting RNA targets. The assay comprisesthe system described by Tuschl et al., 1999 , Genes and Development, 13,3191-3197 and Zamore et al., 2000, Cell, 101, 25-33 adapted for use witha target RNA. A Drosophila extract derived from syncytial blastoderm isused to reconstitute RNAi activity in vitro. Target RNA is generated viain vitro transcription from an appropriate target expressing plasmidusing T7 RNA polymerase or via chemical synthesis as described herein.Sense and antisense siNA strands (for example 20 uM each) are annealedby incubation in buffer (such as 100 mM potassium acetate, 30 mMHEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minute at 90° C.followed by 1 hour at 37° C., then diluted in lysis buffer (for example100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesiumacetate). Annealing can be monitored by gel electrophoresis on anagarose gel in TBE buffer and stained with ethidium bromide. TheDrosophila lysate is prepared using zero to two-hour-old embryos fromOregon R flies collected on yeasted molasses agar that are dechorionatedand lysed. The lysate is centrifuged and the supernatant isolated. Theassay comprises a reaction mixture containing 50% lysate [vol/vol], RNA(10-50 μM final concentration), and 10% [vol/vol] lysis buffercontaining siNA (10 nM final concentration). The reaction mixture alsocontains 10 mM creatine phosphate, 10 ug/ml creatine phosphokinase, 100um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin(Promega), and 100 uM of each amino acid. The final concentration ofpotassium acetate is adjusted to 100 mM. The reactions are pre-assembledon ice and preincubated at 25° C. for 10 minutes before adding RNA, thenincubated at 25° C. for an additional 60 minutes. Reactions are quenchedwith 4 volumes of 1.25× Passive Lysis Buffer (Promega). Target RNAcleavage is assayed by RT-PCR analysis or other methods known in the artand are compared to control reactions in which siNA is omitted from thereaction.

Alternately, internally-labeled target RNA for the assay is prepared byin vitro transcription in the presence of [alpha-³²P] CTP, passed over aG50 Sephadex column by spin chromatography and used as target RNAwithout further purification. Optionally, target RNA is 5′-P-end labeledusing T4 polynucleotide kinase enzyme. Assays are performed as describedabove and target RNA and the specific RNA cleavage products generated byRNAi are visualized on an autoradiograph of a gel. The percentage ofcleavage is determined by PHOSPHOR IMAGER® (autoradiography)quantitation of bands representing intact control RNA or RNA fromcontrol reactions without siNA and the cleavage products generated bythe assay.

In one embodiment, this assay is used to determine target sites in thetarget RNA target for siNA mediated RNAi cleavage, wherein a pluralityof siNA constructs are screened for RNAi mediated cleavage of the targetRNA target, for example, by analyzing the assay reaction byelectrophoresis of labeled target RNA, or by northern blotting, as wellas by other methodology well known in the art.

Example 6 Nucleic Acid Inhibition of Target RNA

siNA molecules targeted to the human target RNA are designed andsynthesized as described above. These nucleic acid molecules can betested for cleavage activity in vivo, for example, using the followingprocedure.

Two formats are used to test the efficacy of siNAs targeting target.First, the reagents are tested in cell culture to determine the extentof RNA and protein inhibition. siNA reagents are selected against thetarget as described herein. RNA inhibition is measured after delivery ofthese reagents by a suitable transfection agent to cells. Relativeamounts of target RNA are measured versus actin using real-time PCRmonitoring of amplification (e.g., ABI 7700 TAQMAN®). A comparison ismade to a mixture of oligonucleotide sequences made to unrelated targetsor to a randomized siNA control with the same overall length andchemistry, but randomly substituted at each position. Primary andsecondary lead reagents are chosen for the target and optimizationperformed. After an optimal transfection agent concentration is chosen,a RNA time-course of inhibition is performed with the lead siNAmolecule. In addition, a cell-plating format can be used to determineRNA inhibition.

Delivery of siNA to Cells

Cells are seeded, for example, at 1×10⁵ cells per well of a six-welldish in EGM-2 (BioWhittaker) the day before transfection. FormulatedsiNA compositions are complexed in EGM basal media (Bio Whittaker) at37° C. for 30 minutes in polystyrene tubes. Following vortexing, thecomplexed formulated siNA composition is added to each well andincubated for the times indicated. For initial optimization experiments,cells are seeded, for example, at 1×10³ in 96 well plates and siNAcomplex added as described. Efficiency of delivery of siNA to cells isdetermined using a fluorescent siNA complexed with lipid. Cells in6-well dishes are incubated with siNA for 24 hours, rinsed with PBS andfixed in 2% paraformaldehyde for 15 minutes at room temperature. Uptakeof siNA is visualized using a fluorescent microscope.

TAQMAN® (Real-Time PCR Monitoring of Amplification) and LightcyclerQuantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example,using Qiagen RNA purification kits for 6-well or Rneasy extraction kitsfor 96-well assays. For TAQMAN® analysis (real-time PCR monitoring ofamplification), dual-labeled probes are synthesized with the reporterdye, FAM or JOE, covalently linked at the 5′-end and the quencher dyeTAMRA conjugated to the 3′-end. One-step RT-PCR amplifications areperformed on, for example, an ABI PRISM 7700 Sequence Detector using 50μl reactions consisting of 10 μl total RNA, 100 nM forward primer, 900nM reverse primer, 100 nM probe, 1×TaqMan PCR reaction buffer(PE-Applied Biosystems), 5.5 mM MgCl₂, 300 μM each dATP, dCTP, dGTP, anddTTP, 10U RNase Inhibitor (Promega), 1.25U AMPLITAQ GOLD® (DNApolymerase) (PE-Applied Biosystems) and 10U M-MLV Reverse Transcriptase(Promega). The thermal cycling conditions can consist of 30 minutes at48° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95°C. and 1 minute at 60° C. Quantitation of mRNA levels is determinedrelative to standards generated from serially diluted total cellular RNA(300, 100, 33, 11 ng/rxn) and normalizing to β-actin or GAPDH mRNA inparallel TAQMAN® reactions (real-time PCR monitoring of amplification).For each gene of interest an upper and lower primer and a fluorescentlylabeled probe are designed. Real time incorporation of SYBR Green I dyeinto a specific PCR product can be measured in glass capillary tubesusing a lightcyler. A standard curve is generated for each primer pairusing control cRNA. Values are represented as relative expression toGAPDH in each sample.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparationtechnique (see for example Andrews and Faller, 1991, Nucleic AcidsResearch, 19, 2499). Protein extracts from supernatants are prepared,for example using TCA precipitation. An equal volume of 20% TCA is addedto the cell supernatant, incubated on ice for 1 hour and pelleted bycentrifugation for 5 minutes. Pellets are washed in acetone, dried andresuspended in water. Cellular protein extracts are run on a 10%Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatantextracts) polyacrylamide gel and transferred onto nitro-cellulosemembranes. Non-specific binding can be blocked by incubation, forexample, with 5% non-fat milk for 1 hour followed by primary antibodyfor 16 hour at 4° C. Following washes, the secondary antibody isapplied, for example (1:10,000 dilution) for 1 hour at room temperatureand the signal detected with SuperSignal reagent (Pierce).

Example 7 Evaluation of Serum Stability of Formulated siNA Compositions

As discussed herein, one way to determine the transfection or deliveryefficiency of the formulated lipid composition is to determine thestability of the formulated composition in serum in vitro. Relativeturbidity measurement can be used to determine the in vitro serumstability of the formulated siNA compositions.

Turbidity measurements were employed to monitor the serum stability oflipid particle formulations L065, F2, L051, and L073 (see FIGS. 14 and15 for the lipid formulations of L051 and L073). The lipid formulationof L065 comprises cationic lipid CpLinDMA, neutral lipid DSPC,cholesterol, and 2 kPEG-DMG. The lipid formulation F2 comprises DODAP.The absorbance of formulated siNA compositions (0.1 mg/ml) in theabsence and presence of 50% serum was measured at 500 nm with acorresponding amount of serum alone as a reference by using SpectraMax®Plus384 microplate spectrophotometer from Molecular Devices (Sunnyvale,Calif.). The formulations were incubated at 37° C. and analyzed at 2min, 5 min, 10 min, 20 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 7 h and 24h. Relative turbidity was determined by dividing the sample turbidity bythe turbidity of 2 min formulated siNA compositions incubated in 50%serum. A formulation or composition is stable in serum if the relativeturbidity remains constant around 1.0 over time. As shown in FIG. 17,formulated siNA compositions L065, L051, and L073 are serum-stable lipidnanoparticle compositions. As shown in FIG. 39, formulated siNAcompositions L077, L080, L082 and L083, are serum-stable lipidnanoparticle compositions.

Example 8 Evaluation of pH-Dependent Phase Transition of Formulated siNACompositions

Additionally, the transfection or delivery efficiency of the formulatedlipid composition can be determined by determining the pH-dependentphase transition of the formulated composition in vitro. Relativeturbidity measurement can be used to determine the pH-dependent phasetransition of formulated siNA compositions in vitro.

Turbidity measurement was employed to monitor the phase transition offormulated siNA compositions L065, L051, F2, L073, and L069. Theabsorbance of lipid particle formulations (0.1 mg/ml) in 0.1 M phosphatebuffer with pH at 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5and 9.0 was measured at 350 nm with a corresponding amount of bufferalone as a reference by using SpectraMax® Plus384 microplatespectrophotometer from Molecular Devices (Sunnyvale, Calif.). This assaymeasures the relative light scattering of the formulations at variouspH. The lamellar structure (i.e., serum stable structure) havingrelatively bigger particle size is expected to scatter more light thanthe corresponding inverted hexagonal structure. The samples wereincubated at 37° C. and analyzed at 2 min, 5 min, 10 min, 30 min, and 2h. Relative turbidity was determined by dividing the sample turbidity bythe turbidity of 2 min formulated siNA compositions incubated inphosphate buffer at pH 7.5. A formulation or composition undergoespH-dependent phase transition if there is a change in the relativeturbidity when measured between pH 7.5-pH 5.0. As shown in FIG. 18,formulated siNA compositions L051 and L073 undergo pH-dependent phasetransition at pH 6.5-pH 5.0. As shown in FIG. 19, formulated siNAcomposition L069 undergoes pH-dependent phase transition at pH 6.5-pH5.0. As shown in FIG. 40, formulated siNA compositions L077, L080, L082,and L083 undergo pH-dependent phase transition at pH 6.5-pH 5.0.

Example 9 Evaluation of Formulated siNA Compositions in Models ofChronic HBV Infection

In Vitro Analysis of siNA Nanoparticle Activity

Hep G2 cells were grown in EMEM (Cellgro Cat#10-010-CV) withnon-essential amino acids, sodium pyruvate (90%), and 10% fetal bovineserum (HyClone Cat#SH30070.03). Replication competent cDNA was generatedby the excision and re-ligation of the HBV genomic sequences from thepsHBV-1 vector. HepG2 cells were plated (3×10⁴ cells/well) in 96-wellmicrotiter plates and incubated overnight. A cationic lipid/DNA complexwas formed containing (at final concentrations) cationic lipid (11-15μg/mL), and re-ligated psHBV-1 (4.5 μg/mL) in growth media. Following a15 min incubation at 37° C., 20 μL of the complex was added to theplated HepG2 cells in 80 μL of growth media minus antibiotics. After 7.5hours at 37° C., the media was then removed, the cells rinsed once withmedia, and 100 μL of fresh media was added to each well. 50 μL of thesiNA nanoparticle formulation (see Example 9 for formulation details)(diluted into media at a 3× concentration) was added per well, with 3replicate wells per concentration. The cells were incubated for 4 days,the media was then removed, and assayed for HBsAg levels. FIG. 21 showslevel of HBsAg from formulated (Formulation L051, Table IV) active siNAtreated cells compared to untreated or negative control treated cells.FIG. 22 shows level of HBsAg from formulated (Formulations L053 andL054, Table IV) active siNA treated cells compared to untreated ornegative control treated cells. FIG. 23 shows level of HBsAg fromformulated (Formulation L051, Table IV) active siNA treated cellscompared to untreated or negative control treated cells. FIG. 36 showslevel of HBsAg from formulated (Formulations L083 and L084, Table IV)active siNA treated cells compared to untreated or negative controltreated cells. FIG. 37 shows level of HBsAg from formulated (FormulationL077, Table IV) active siNA treated cells compared to untreated ornegative control treated cells. FIG. 38 shows level of HBsAg fromformulated (Formulation L080, Table IV) active siNA treated cellscompared to untreated or negative control treated cells. In thesestudies, a dose dependent reduction in HBsAg levels was observed in theactive formulated siNA treated cells using nanoparticle formulationsL051, L053, and L054, while no reduction is observed in the negativecontrol treated cells. This result indicates that the formulated siNAcompositions are able to enter the cells, and effectively engage thecellular RNAi machinery to inhibit viral gene expression.

Analysis of Formulated siRNA Activity in a Mouse Model of HBVReplication

To assess the activity of chemically stabilized siNA nanoparticle (seeExample 9 for formulation details) compositions against HBV, systemicdosing of the formulated siNA composition (Formulation L051, Table IV)was performed following hydrodynamic injection (HDI) of the HBV vectorin mouse strain NOD.CB17-Prkdc^(scid)/J (Jackson Laboratory, Bar Harbor,Me.). Female mice were 5-6 weeks of age and approximately 20 grams atthe time of the study. The HBV vector used, pWTD, is a head-to-taildimer of the complete HBV genome. For a 20-gram mouse, a total injectionof 1.6 ml containing pWTD in saline, was injected into the tail veinwithin 5 seconds. A total of 0.3 μg of the HBV vector was injected permouse. In order to allow recovery of the liver from the disruptioncaused by HDI, dosing of the formulated siNA compositions were started 6days post-HDI. Encapsulated active or negative control siRNA wereadministered at 3 mg/kg/day for three days via standard IV injection.Groups (N=5) of animals were sacrificed at 3 and 7 days following thelast dose, and the levels of serum HBV DNA and HBsAg were measured. HBVDNA titers were determined by quantitative real-time PCR and expressedas mean log 10 copies/ml (±SEM). The serum HBsAg levels were assayed byELISA and expressed as mean log 10 μg/ml (±SEM). Significant reductionsin serum HBV DNA (FIGS. 24 and 41) and HBsAg (FIGS. 25, 36, 37, and 38)were observed at both the 3 and 7-day time points in the activeformulated siNA composition treated groups as compared to both the PBSand negative control groups.

Materials and Methods Oligonucleotide Synthesis and Characterization

All RNAs were synthesized as described herein. Complementary strandswere annealed in PBS, desalted and lyophilized. The sequences of theactive site 263 HBV siNAs are shown in FIG. 20. The modified siNAs usedin vivo are termed HBV263M and HBV1583M, while versions containingunmodified ribonucleotides and inverted abasic terminal caps are calledHBV263R and HBV1583R. Some pharmacokinetic studies were done with siNAtargeting two other sites, HBV1580M and HBV1580R.

The siNA sequences for HCV irrelevant control are:

sense strand: 5′ B-cuGAuAGGGuGcuuGcGAGTT-B 3′ (SEQ ID NO: 1) antisensestrand: 5′ CUC GcAAGcAcccuAucAGTsT 3′ (SEQ ID NO: 2)

(where lower case=2′-deoxy-2′-fluoro; Upper Case italic=2′-deoxy; UpperCase underline=2′-O-methyl; Upper Case Bold=ribonucleotide; T=thymidine;B=inverted deoxyabasic; and s=phosphorothioate)

The inverted control sequences are inverted from 5′ to 3′.

HBsAg ELISA Assay

Levels of HBsAg were determined using the Genetic Systems/Bio-Rad(Richmond, Va.) UBsAg ELISA kit, as per the manufacturer's instructions.The absorbance of cells not transfected with the HBV vector was used asbackground for the assay, and thus subtracted from the experimentalsample values.

HBV DNA Analysis

Viral DNA was extracted from 50 μL mouse serum using QIAmp 96 DNA Bloodkit (Qiagen, Valencia, Calif.), according to manufacture's instructions.HBV DNA levels were analyzed using an ABI Prism 7000 sequence detector(Applied Biosystems, Foster City, Calif.). Quantitative real time PCRwas carried out using the following primer and probe sequences: forwardprimer 5′-CCTGTATTCCCATCCCATCGT (SEQ ID NO: 3, HBV nucleotide2006-2026), reverse primer 5′-TGAGCCAAGAGAAACGGACTG (SEQ ID NO: 4, HBVnucleotide 2063-2083) and probe FAM 5′-TTCGCA AAATACCTATGGGAGTGGGCC (SEQID NO: 5, HBV nucleotide 2035-2062). The psHBV-1 vector, containing thefull length HBV genome, was used as a standard curve to calculate HBVcopies per mL of serum.

Example 10 Evaluation of Formulated siNA Compositions in an In Vitro HCVReplicon Model of HCV Infection

An HCV replicon system was used to test the efficacy of siNAs targetingHCV RNA. The reagents were tested in cell culture using Huh7 cells (seefor example Randall et al., 2003, PNAS USA, 100, 235-240) to determinethe extent of RNA inhibition. siNA were selected against the HCV targetas described herein. The active siNA sequences for HCV site 304 are asfollows: sense strand: (SEQ ID NO: 1); antisense strand: (SEQ ID NO: 2)(these were used as inactive sequences in Example 8 above). The siNAinactive control sequences used in the study target HBV site 263 and areas follows: sense strand: (SEQ ID NO: 6); antisense strand: (SEQ ID NO:7), (these were used as active sequences in Example 8 above). The activeand inactive siNAs were formulated as Formulation L051, L053, or L054 asdescribed in Example 9 above. Huh7 cells, containing the stablytransfected Clone A HCV subgenomic replicon (Apath, LLC, St. Louis,Mo.), were grown in DMEM (Invitrogen catalog #11965-118) with 5 mls of100× (10 mM) Non-Essential Amino Acids (Invitrogen catalog #11140-050),5 uL of 200 mM Glutamine (Cellgro catalog#25-005-C1), 50 uL of heatinactivated Fetal Bovine Serum (Invitrogen catalog #26140-079) and 1mg/mLG418 (Invitrogen catalog#11811-023). For transfection with siNAformulations, cells are plated at 9,800 cells per well into a 96-wellCoStar tissue culture plate using DMEM with NEAA and 10% FBS, (noantibiotics). After 20-24 hours, cells were transfected with formulatedsiNA for a final concentration of 1-25 nM. After incubating for 3 days,the cells were lysed and RNA extracted using the RNaqueous-96 kit(Ambion Cat#1920) as per the manufacturers instructions. FIG. 26 showslevel of HCV RNA from formulated (Formulation L051, Table IV) activesiNA treated cells compared to untreated or negative control treatedcells. FIG. 27 shows level of HCV RNA from formulated (Formulations L053and L054, Table IV) active siNA treated cells compared to untreated ornegative control treated cells. In these studies, a dose dependentreduction in HCV RNA levels was observed in the active formulated siNAtreated cells using formulations L051, L053, and L054, while noreduction is observed in the negative control treated cells. This resultindicates that the formulated siNA compositions are able to enter thecells, and effectively inhibit viral gene expression.

Example 11 Lung Distribution of Unformulated and Formulated siNA afterIntratracheal Dosing

To determine the efficiency of delivery of siNA molecules to the lung,unformulated siRNA (naked), cholesterol conjugated siNA, or siRNA informulated molecular compositions (T018.1 and T019.1) were administeredvia the trachea to the lungs of mice. Unformulated siNA comprises nakednucleic acid. Cholesterol conjugated siNA comprises siNA linked tocholesterol. Formulated molecular compositions T018.1 and T019.1comprise siNA formulated with DOcarbDAP, DSPC, cholesterol and PEG-DMG,and DODMA, DSPC, cholesterol and PEG-DMG, respectively. Groups of threefemale C57 Bl/6 mice were placed under anesthesia with ketamine andxylazine. Filtered dosing solutions were administered via the trachea at1.0 mg/kg duplexed siRNA, using a Penncentury model #1A-1C microsprayerand a Penncentury model #FMJ250 syringe to aerosolize the siRNA (TGFβsite 1264 stabilization chemistry 7/8) directly into the lungs. Animalswere dosed with unformulated siNA, cholesterol-conjugated siNA or siNAin formulated molecular compositions. At 1, 24 or 72 hours after dosing,the animals were euthanized, exsanguinated and perfused with sterileveterinary grade saline via the heart. The lungs were removed, placed ina pre-weighed homogenization tube and frozen on dry ice. Lung weightswere determined by subtraction after weighing the tubes plus lungs.Levels of siNA in the lung tissue were determined using a hybridizationassay. FIG. 28, shows the levels of siNA in lung tissue after directdosing of (i) unformulated siNA, (ii) cholesterol conjugated siNA or(iii) siNA in formulated molecular compositions T018.1 or T019.1. Halflives of exposure in lung tissue were 3-4 hours for the unformulatedsiNA, 9 hours for the cholesterol conjugated siNA and 37-39 hours forthe siNA in formulated molecular compositions T018.1 or T019.1.

Example 12 Efficient Transfection of Various Cell Lines Using siNA LNPFormulations of the Invention

The transfection efficacy of LNP formulations of the invention wasdetermined in various cell lines, including 6.12 spleen, Raw 264.7tumor, MM14Lu, NIH 3T3, D10.G4.1 Th2 helper, and lung primary macrophagecells by targeting endogenous MAP Kinase 14 (p38) gene expression. Apotent lead siNA against MapK4 (p38a) was selected by in vitro screeningusing Lipofectamine 2000 (LF2K) as the delivery agent. The sense strandsequence of this siNA comprised 5′-B cuGGuAcAGAccAuAuuGATT B-3′ (SEQ IDNO: 6) and the antisense strand sequence comprised5′-UCAAuAuGGucuGuAccAGTsT-3′ (SEQ ID NO: 7), where lowercase=2′-deoxy-2′-fluoro; Upper Case italic=2′-deoxy; Upper Caseunderline=2′-O-methyl; Upper Case Bold=ribonucleotide; T=thymidine;B=inverted deoxyabasic; and s=phosphorothioate).

Proprietary MapK14 targeted LNPs were screened and compared to LF2K anda LNP control containing an inactive siNA in cultured cells.Furthermore, lead LNPs were tested in a dose response method todetermine IC50 values. Results are summarized in Table V. FIG. 41 showsefficacy data for LNP 58 and LNP 98 formulations targeting MapK14 site1033 in RAW 264.7 mouse macrophage cells. FIG. 42 shows efficacy datafor LNP 98 formulations targeting MapK14 site 1033 in MM14.Lu normalmouse lung cells. FIG. 43 shows efficacy data for LNP 54, LNP 97, andLNP 98 formulations targeting MapK14 site 1033 in 6.12 B lymphocytecells. FIG. 44 shows efficacy data for LNP 98 formulations targetingMapK14 site 1033 in NIH 3T3 cells. FIG. 45 shows the dose-dependentreduction of MapK14 RNA via MapK14 LNP 54 and LNP 98 formulated siNAs inRAW 264.7 cells. FIG. 46 shows the dose-dependent reduction of MapK14RNA via MapK14 LNP 98 formulated siNAs in MM14.Lu cells. FIG. 47 showsthe dose-dependent reduction of MapK14 RNA via MapK14 LNP 97 and LNP 98formulated siNAs in 6.12 B cells. FIG. 48 shows the dose-dependentreduction of MapK14 RNA via MapK14 LNP 98 formulated siNAs in NIH 3T3cells.

LF2K Transfection Method:

The following procedure was used for LF2K transfection. After 20-24hours, cells were transfected using 0.25 or 0.35 uL Lipofectamine2000/well and 0.15 or 0.25 uL/well, complexed with 25 nM siNA.Lipofectamine 2000 was mixed with OptiMEM and allowed to sit for atleast 5 minutes. For 0.25 uL transfections, 1 uL of LF2K was mixed with99 uL OptiMEM for each complex. For 0.35 uL transfections, 1.4 uL ofLF2K was mixed with 98.6 uL OptiMEM for each complex. For 0.15 uLtransfections, 0.60 uL of SilentFect was mixed with 99.4 uL OptiMEM foreach complex. For 0.30 uL transfections, 1.2 uL of SilentFect was mixedwith 98.2 uL OptiMEM for each complex. The siNA was added to amicrotitre tube (BioRad #223-9395) and OptiMEM was then added to make100 uL total volume to be used in 4 wells. 100 uL of the Lipofectamine2000/OptiMEM mixture was added and the tube was vortexed on medium speedfor 10 seconds and allowed to sit at room temperature for 20 minutes.The tube was vortexed quickly and 50 uL was added per well, whichcontained 100 uL media. RNA from treated cells was isolated at 24, 48,72, and 96 hours.

LNP Transfection Method:

The following procedure was used for LNP transfection. Cells were platedto the desired concentration in 100 uL of complete growth medium in96-well plates, ranging from 5,000-30,000 cells/well. After 24 hours,the cells were transfected by diluting a 5× concentration of LNP incomplete growth medium onto the cells, (25 uL of 5× results in a finalconcentration of 1×). RNA from treated cells was isolated at 24, 48, 72,and 96 hours.

Example 13 Reduction of Airway Hyper-Responsiveness in a Mouse Model ofAsthma

An OVA induced airway hyper-responsiveness model was used to evaluateLNP formulated siNA molecules targeting interleukin 4R (IL-4R alpha) forefficacy in reducing airway hyper-responsiveness. The sense strandsequence of the active siNA targeting IL-4R alpha used in this studycomprised 5′-B ucAGcAuuAccAAGAuuAATT B-3′ (SEQ ID NO: 8) and theantisense strand sequence comprised 5′-UUAAucuuGGuAAuGcuGATsT-3′ (SEQ IDNO: 9), where lower case=2′-deoxy-2′-fluoro; Upper Case italic=2′-deoxy;Upper Case underline=2′-O-methyl; Upper Case Bold=ribonucleotide;T=thymidine; B=inverted deoxyabasic; and s=phosphorothioate).On Day 0and 7, the animals were immunized by intraperitoneal injection of 0.4mg/mL OVA/saline solution mixed in an equal volume of Imject Alum for afinal injection solution of 0.2 mg/mL (100 uL/mouse). LNP-51 formulatedIL-4R-alpha Site 1111 siNA (see U.S. Ser. No. 11/001,347, incorporatedby reference herein), prepared in PBS (w/o Ca2+, Mg2+), or irrelevantcontrol was delivered by intratracheal dosing qd (once every day)beginning on Day 17 and ending on Day 26 for a total of 10 doses. Micewere aerosol challenged with OVA (1.5% in saline) for 30 minutes on days24, 25 and 26 using the Pari LC aerosol nebulizer. Animals were allowedto rest for 24 hours prior to airway function analysis. On Day 28 airwayresponsiveness was assessed after challenge with aerosolizedmethacholine using the Buxco Whole Body Plethysmograph. Aftermethacholine challenge, animals were euthanized. A tracheotomy wasperformed, and the lungs were lavaged with 0.5 mL of saline twice. Lunglavage was performed while massaging the animal's chest and all lavagefluid were collected and placed on ice. A cytospin preparation wasperformed to collect the cells from the BAL fluid for differential cellcounts. Results are shown in FIG. 49, which clearly demonstrates theactivity of the formulated siNA in a dose response (0.01, 0.1, and 1mg/kg) compared to the LNP vehicle alone and untreated (naïve) animals.

Example 14 Efficient Reduction in Human Huntingtin (htt) Gene ExpressionIn Vivo Using LNP Formulated siNA

Huntington's disease (HD) is a dominant neurodegenerative disordercaused by an expansion in the polyglutamine (polyQ) tract of thehuntingtin (htt) protein. PolyQ expansion in htt induces cortical andstriatal neuron cell less, and the formation of htt-containingaggregates within brain cells. HD patients have progressive psychiatric,cognitive and motor dysfunction and premature death. Early work in mousemodels has demonstrated that reduction of mutant protein after the onsetof disease phenotypes could improve motor dysfunction and reducehtt-aggregate burden. Thus, reduction of mutant htt in patient brain mayimprove the disease.

Recent work has shown that reduction of mutant htt in a mouse model ofHD, using a viral vector expressing short interfering RNAs (siRNAs),protected the animal from the onset of behavioral and neuropathologicalhallmarks of the disease (see Harper et al., 2005, PNAS USA, 102:5820-5). This study was utilized to determine whether delivery ofsynthetic siNAs directly to the brain by nonviral methods could besimilarly effective. This approach has many advantages, including theability to modify dosing regimines. Chemically modified siNA, sensestrand having sequence 5′-B AccGuGuGAAucAuuGucuTT B-3′ (SEQ ID NO:10)and antisense strand 5′-AGAcAAuGAuucAcAcGGuTsT-3′ (SEQ ID NO: 11)encapsulated in lipid nanoparticles (LNP) formulations LNP-061, LNP-098,and LNP-101 (see Table IV) were utilized in this study. In thesesequences, lower case stands for 2′-deoxy-2′-fluoro, Upper Case standsfor ribonucleotides, underline Upper Case stands for 2′-O-methylnucleotides, T is thymidine, s is phosphorothioate, and B is inverteddeoxy abasic. The siNA duplexes encapsulated in the various LNPformulations were screened for their ability to silence full-length httin vitro, followed by testing in vivo. Using Alzet osmotic pumps, siNAsencapsulated in LNPs were infused into the lateral ventrical or striatumfor 7 or 14 days, respectively, at concentrations ranging from 0.1 to 1mg/ml (total dose ranging from 8.4 to 84 μg). An impressive 80%reduction in htt mRNA levels was observed in animals treated withLNP-061 and LNP-098 formulated siNA as determined by QPCR compared toscrambled control sequences, or naïve brain. Results are shown in FIG.50.

Example 15 Potentiated RNAi Efficacy of LNP Formulated siNAs by Additionof Carrier LNP Containing Non Targeting Nucleic Acid

As shown in the various embodiments and examples herein, Applicant hasdeveloped several lipid nanoparticle (LNP) formulations that efficientlyencapsulate biologically active molecules such as short interferingnucleic acid (siNA) molecules. The injection of these LNP formulatedsiNAs by intravenous route in mouse results in efficient delivery totarget organs like liver and results in potent and specific knockdown ofintended targets (see Example 9 above). Local delivery of LNP formulatedsiNA molecules also results in efficient delivery to target organs suchas lung and CNS and results in potent and specific knockdown of intendedtargets (see Examples 13 and 14 above). These formulated siNAs alsotransfect the tissue culture cells efficiently and show specific RNAiactivity (see Example 12). Applicant describes herein a generalmethodology for potentiating the delivery of biologically activemolecules using lipid based delivery vehicles by utilizing formulatedLNP siNA compositions in conjunction with non-targeted polynucleotidecarrier molecules of the invention.

Potentiated In Vitro Activity of LNP Formulated Active siNA Compositionsin Conduction with LNP Formulated Inactive Carrier Molecules

To calculate the IC50 of LNP formulated HBV263 siNA, dose responsestudies were carried out in HepG2 cells stably expressing Hepatitis Bvirus (HBV) as described in Example 9 herein. The concentration seriesof L124 (Table IV) LNP formulated HBV263 siNA was prepared in twodifferent ways. In the first series, active siNA was diluted directly into tissue culture media to achieve the required concentration. In thesecond series, active formulated siNA was diluted with carrier LNPencapsulated control siNA (HBV263 inverted sequence) to keep the finalconcentration of total siNA concentration the same in each sample (seeexperimental section for details). As shown in FIG. 51, the activeformulated siNA series prepared in the presence of the formulatedcarrier control siNA showed potentiated RNAi activity compared to theactive formulated siNA series that does not contain the formulatedcarrier control siRNA. This experiment suggested that the presence ofcarrier LNP formulated control siNA improved the RNAi activity of LNPformulated active siNA, for example through a mechanism of increasedintracellular delivery of the active siNA species.

Potentiated In Vivo Activity of LNP Formulated Active siNA Compositionsin Conduction with LNP Formulated Inactive Carrier Molecules

Applicant carried out a similar experiment in an in vivo setting byinjecting LNP formulated active siNA targeting Sjogren syndrome antigenB (SSB) RNA. Male Balb/C mice were dosed intravenously once daily forthree days with 0.1, 0.3 and 3 mg/kg of L124 (Table IV) formulatedSSB291 siNA (corresponding total lipid doses are approximately 9, 30 and90 mg/kg). SSB291 siNA was diluted in phosphate buffered saline (PBS)alone, or with carrier LNP (L124 formulated HCV316 non targeting siNA)such that the total siNA dose would equal 3 mg/kg (or 90 mg/kg totallipid dose). Three days following the last dose, the animals weresacrificed and livers collected for RNA isolation. Total RNA wasisolated from approximately 100 mg liver tissue using Tri-Reagent(Sigma) according to manufacture's instruction. SSB RNA levels werequantitated and normalized to mouse GAPDH RNA using real timereverse-transcription (RT)-PCR. Relative amounts of both SSB and GAPDHRNA were calculated from a standard curve of total liver RNA collectedfrom a PBS control animal (3-fold serial dilutions from 300 ng-1 ng RNAper reaction). The data is expressed as a ratio of SSB to GAPDH RNA. Asshown in FIG. 52, the presence of carrier LNP significantly improved theactivity of SSB291 siRNA. When active SSB291 siNA is dosed at 0.3 mg/kg,target knockdown increased from 45% without carrier LNP to 75% withcarrier LNP. It is clear from these experiments that carrier LNPpotentiates RNAi activity of the active siNA, for example viaestablishing or maintaining a critical mass of total lipid formulationto be delivered intracellularly.

To explore the role of the inactive duplex siNA as carrier (duplex siNAhaving no complementarity to the RNA target), a comparison was made withan inactive single stranded (SS) polynucleotide (single strand having nocomplementarity to the RNA target). The modified sense strand of theHBV263 siNA duplex was formulated with L124 and a assayed along with theformulated HCV316 siNA duplex. The SSB291 siNA duplex was dosed at 0.3mg/kg alone, or in the presence of carrier LNP at a total dose of 3mg/kg as described above. Total liver RNA was isolated and analyzed forSSB RNA as described above. As shown in FIG. 53, SSB291 showed 37%knockdown of target RNA but when supplied with carrier LNP containingeither inactive duplex or single stand polynucleotides, the knockdownefficiency improved to ˜79% and 70%, respectively. The carrier LNP onits own showed no significant knockdown of SSB target. Thus, carrier LNPcan be made with either single stranded or double stranded and stillsignificantly improve the RNAi activity of active siNA.

Not wishing to be bound to any particular theory, the potentiation inthe biologic activity (e.g., RNAi activity) of delivery vehicleassociated biologically active molecule(s) (e.g., active LNP formulatedsiNA) seen in presence of one or more carrier molecules (e.g., inactiveLNP formulated polynucleotides) can be explained by one or moretheories. For example, a critical amount of delivery vehicle associatedbiologically active molecules (e.g., LNP encapsulated active siNA) canbe required for effective delivery of the biologically active molecules(e.g., active siNA) in cellular compartments where biological activitytakes place (e.g., for siNA, where the siNA is available for formationof the RISC complex). One hypothesis is that a certain amount orconcentration of the delivery vehicle and the biologically activemolecule (e.g., LNP formulated active siNA) is needed for efficientrelease of the biologically active molecules (e.g., active siNA) fromendosomes. It is also possible that there are cells or cellularcompartments that efficiently take up the delivery vehicle and its cargo(e.g., LNP formulated active siNA) but that do not make it available forbiologic activity to take place (e.g., in the case of siNA, RISC loadingand Ago1/Ago2 activity). In such instance, at low concentrations offormulated biologically active molecule (e.g., LNP formulated siNA), itwould be difficult for the biologically active molecule(s) to reachcells or cellular compartments where biologic activity can take place(e.g., where the RNAi machinery resides). For example, by increasing thecritical mass of LNP formulations with carrier LNP, it will allow evensmall amounts of active LNP to reach cellular compartments where theyare utilized in RNAi pathway. The carrier phenomenon can have severaladvantages. The effective amount of biologically active molecule(s)(e.g., active siNA) needed to induce RNAi activity can be dramaticallyreduced by carrier molecules (e.g., inactive polynucleotides). The useof carrier molecule(s) also provides the opportunity to use a mixture ofbiologically active molecules against the same or differingintracellular targets so that a critical amount of the biologicallyactive molecule can be achieved for effective biologic activity whilereducing the concentration of individual biologically active molecule(s)that are required for such biologic activity.

Materials and Methods (Sequences are Shown in Table IX)

In Vitro Activity of siNAs in Cell CultureTransfection Protocol for HepG2 siNA Studies:

Cells are grown in EMEM (Cellgro Cat#10-010-CV) with non-essential aminoacids, sodium pyruvate, glutamine (90%), and 10% fetal bovine serum(HyClone Cat#SH30070.03).

Plating Cells:

For transfection, cells are plated in 80 uL growth media at 42,000 cellsper well (525,000/mL) into 96-well tissue culture plate (CostarCat#3596). Cells are transfected 16-24 hours later.

Testing HBV Formulations: For Vector Only Transfection:

Transfection complex for 1 plate of 60 wells 80 uL plasmid (pSV-HBV-1)1,430 uL media80 uL 168 (1 mg/mL)Vortex at 4.5 for 5 seconds, put into incubator for 15-20 minutes,vortexAliquot to Biorad microtitre tubes and add 20 uL of this per wellAllow transfection to occur for 6-7 hours in the incubator.

For Formulation Transfections:

Aspirate media, wash once with 100 uL media, then add 100 uL fresh mediaTo each well add 50 uL of the 3× formulation L124.1.2.7Au diluted inmedia or 30 nM Inactive or inverted HBVi L12.1.2.3Cu.Incubate 3 days.Run ELISA on supernatant according to directions. (Genetic Systems HBsAgEIA 3.0)

Example 16 Preparation of Cationic Lipids of the Invention (see FIGS.29A and 29B for Synthetic Schemes) Cholest-5-en-3β-tosylate (2)

Cholesterol (1, 25.0 g, 64.7 mmol) was weighed into a 1 L round bottomedflask with a stir bar. The flask was charged with pyridine (250 mL),septum sealed and flushed with argon. Toluenesulfonyl chloride (25.0 g,131 mmol) was weighed into a 100 mL round bottomed flask, which was thensealed and charged with pyridine. The toluenesulfonyl chloride solutionwas then transferred, via syringe, to the stirring cholesterol solution,which was allowed to stir overnight. The bulk of pyridine was removed invacuo and the resulting solids were suspended in methanol (300 mL) andstirred for 3 hours, until the solids were broken up into a uniformsuspension. The resultant suspension was filtered and the solids werewashed with acetonitrile and dried under high vacuum to afford 31.8 g(91%) of a white powder (see for example Davis, S. C.; Szoka, F. C., Jr.Bioconjugate Chem. 1998, 9, 783).

Cholest-5-en-3β-oxybutan-4-ol (3a)

Cholest-5-ene-3β-tosylate (20.0 g, 37.0 mmol) was weighed into a 500 mLround bottomed flask with a stir bar. The flask was charged with dioxane(300 mL) and 1,4-butanediol (65.7 mL, 20 equiv.). The flask was fittedwith a reflux condenser and the mixture was brought to reflux overnight.The reaction was cooled and concentrated in vacuo. The reaction mixturewas suspended in water (400 mL). The solution was extracted withmethylene chloride (3×200 mL). The organic phases were combined andwashed with water (2×200), dried over magnesium sulfate, filtered andthe solvent removed. The resultant oil/wax was further purified viacolumn chromatography (15% Acetone/Hexanes) to afford 13.41 g (79%) of acolorless wax.

Cholest-5-en-3β-oxypent-3-oxa-an-5-ol (3b)

This compound was prepared similarly to cholest-5-en-3β-oxybutan-4-ol.Cholest-5-ene-3,3-tosylate (5.0 g, 9.2 mmol) was weighed into a 500 mLround bottomed flask with a stir bar. The flask was charged with dioxane(150 mL) and diethylene glycol (22 mL, 25 equiv.). The flask was fittedwith a reflux condenser and the mixture was brought to reflux overnight.The reaction was cooled and concentrated. The reaction mixture wassuspended in water (500 mL). The solution was extracted with methylenechloride (3×200 mL). The organic phases were combined and washed withwater (2×200 mL), dried over magnesium sulfate, filtered and the solventremoved. The resultant oil/wax was further purified via columnchromatography (25% EtOAc/Hexanes) to afford 3.60 g (82%) of colorlessoil (see for example Davis, S. C.; Szoka, F. C., Jr. Bioconjugate Chem.1998, 9, 783).

Cholest-5-en-3β-oxybutan-4-mesylate (4a)

Cholest-5-en-3β-oxybutan-4-ol (12.45 g, 27.14 mmol) was weighed into a500 mL round bottomed flask with a stir bar. The flask was sealed,flushed with argon, charged with methylene chloride (100 mL) andtriethylamine (5.67 mL, 1.5 equiv.) and cooled to 0° C. Methanesulfonylchloride (3.15 mL, 1.5 equiv.) was measured in a PP syringe and addedslowly to the stirring reaction mixture. The reaction was allowed tostir for 1 hr at 0° C. when TLC analysis (7.5% EtOAc/Hexanes) showedthat the reaction was complete. The reaction mixture was diluted withmethylene chloride (100 mL) and washed with saturated bicarbonatesolution (2×200 mL) and brine (1×100 mL). The organic phase was driedover MgSO₄, filtered and concentrated to give 14.45 g (99%) of acolorless wax that was used without further purification.

Cholest-5-en-3β-oxypent-3-oxa-an-5-mesylate (4b)

This compound was prepared similarly toCholest-5-en-3β-oxybutan-4-mesylate.Cholest-5-en-3β-oxypent-3-oxa-an-5-ol (3.60 g, 7.58 mmol) was weighedinto a 500 mL round bottomed flask with a stir bar. The flask wassealed, flushed with argon, charged with methylene chloride (30 mL) andtriethylamine (1.60 mL, 1.5 equiv.) and cooled to 0° C. Methanesulfonylchloride (0.89 mL, 1.5 equiv.) was measured in a PP syringe and addedslowly to the stirring reaction mixture. The reaction was allowed tostir for 1 hr at 0° C. when TLC analysis (10% EtOAc/Hexanes) showed thatthe reaction was complete. The reaction mixture was diluted withmethylene chloride (150 mL) and washed with saturated bicarbonatesolution (2×100 mL) and brine (1×100 mL). The organic phase was driedover MgSO₄, filtered and concentrated to give 4.15 g (99%) of acolorless wax that was used without further purification.

1-(4,4′-Dimethoxytrityloxy)-3-dimethylamino-2-propanol (5)

3-Dimethylamino-1,2-propanediol (6.0 g, 50 mmol) was weighed into a 1 Lround bottomed flask with a stir bar. The flask was sealed, flushed withargon, charged with pyridine and cooled to 0° C. 4,4′-Dimethoxytritylchloride (17.9 g, 1.05 equiv.) was weighed into a 100 mL round bottomedflask, sealed and then dissolved in pyridine (80 mL). The4,4′-dimethoxytrityl chloride solution was transferred to the stirringreaction mixture slowly, using additional fresh pyridine (20 mL) toeffect the transfer of residual 4,4′-dimethoxytrityl chloride. Thereaction was allowed to come to room temperature while stirringovernight. The reaction was concentrated in vacuo and re-dissolved indichloromethane (300 mL). The organic phase was washed with saturatedbicarbonate (2×200 mL) and brine (1×200 mL), dried over MgSO₄, filtered,concentrated and dried under high vacuum to afford 22.19 g of a yellowgum that was used without further purification.

3-Dimethylamino-2-(cholest-5-en-3β-oxybutan-4-oxy)-1-propanol (6a)

1-(4,4′-Dimethoxytrityloxy)-3-Dimethylamino-2-propanol (7.50 g, 17.8mmol) was weighed into a 200 mL round bottomed flask and co-evaporatedwith anhydrous toluene (2×50 mL). A stir bar was added to the flaskwhich was septum sealed, flushed with argon and charged with toluene (60mL). Sodium hydride (1.71 g, 4 equiv.) was added at once and the mixturewas stirred at room temperature for 20 minutes.Cholest-5-en-3β-oxybutan-4-mesylate was dissolved in anhydrous toluene(20 mL) and added to the reaction mixture, via syringe. The flask wasfitted with a reflux condenser with a continuous argon stream and thereaction was heated to reflux overnight. The reaction mixture was cooledto room temperature in a water bath and ethanol was added dropwise untilgas evolution ceased. The reaction mixture was diluted with ethylacetate (300 mL) and washed with aqueous 10% sodium carbonate (2×300mL). The aqueous phases were combined and back extracted with ethylacetate (2×100 mL). The organic phases were combined, dried over MgSO₄,filtered and concentrated to an oil in a 500 mL round bottomed flask.

The flask was fitted with a stir bar, sealed, purged with argon andcharged with dichloroacetic acid solution (3% in DCM, 200 mL).Triethylsilane (14.2 mL, 89 mmol) was added to the mixture and thereaction was allowed to stir overnight. The reaction mixture was dilutedwith DCM (300 mL) and washed with saturated bicarbonate solution (2×200mL). The aqueous phases were combined and back extracted with DCM (2×100mL). The organic phases were combined and dried over MgSO₄, filtered andconcentrated to an oil that was re-dissolved in ethanol (150 mL).Potassium fluoride (10.3 g, 178 mmol) was added to the solution, whichwas then brought to reflux for 1 hr. The mixture was cooled,concentrated in vacuo, re-dissolved in DCM (200 mL), filtered andconcentrated to an oil/crystal mixture. The mixture was re-dissolved ina minimum of DCM and loaded onto a silica gel column which waspre-equilibrated and eluted with 25% EtOAc/Hexanes with 3% TEA to afford4.89 g (49%) of a colorless wax.

3-Dimethylamino-2-(cholest-5-en-3β-oxypent-3-oxa-an-5-oxy)-1-propanol(6b)

This compound was prepared similarly to3-Dimethylamino-2-(Cholest-5-en-3β-oxybutan-4-oxy)-1-propanol.1-(4,4′-Dimethoxytrityloxy)-3-Dimethylamino-2-propanol (2.65 g, 6.31mmol) was weighed into a 200 mL round bottomed flask and co-evaporatedwith anhydrous toluene (2×20 mL). A stir bar was added to the flaskwhich was septum sealed, flushed with argon and charged with toluene (50mL). Sodium hydride (0.61 g, 4 equiv.) was added at once and the mixturewas stirred at room temperature for 20 minutes.Cholest-5-en-3β-oxypent-3-oxa-an-5-mesylate (4.15 g, 7.6 mmol) wasdissolved in anhydrous toluene (10 mL) and added to the reactionmixture, via syringe. The flask was fitted with a reflux condenser witha continuous argon stream and the reaction was heated to refluxovernight. The reaction mixture was cooled to room temperature in awater bath and ethanol was added dropwise until gas evolution ceased.The reaction mixture was diluted with ethyl acetate (200 mL) and washedwith aqueous 10% sodium carbonate (2×200 mL). The aqueous phases werecombined and back extracted with ethyl acetate (2×100 mL). The organicphases were combined, dried over MgSO₄, filtered and concentrated to anoil in a 500 mL round bottomed flask.

The flask was fitted with a stir bar, sealed, purged with argon andcharged with dichloroacetic acid solution (3% in DCM, 150 mL).Triethylsilane (4.03 mL, 25.2 mmol) was added to the mixture and thereaction was allowed to stir for 4 hours. The reaction mixture wasdiluted with DCM (100 mL) and washed with saturated bicarbonate solution(2×200 mL). The aqueous phases were combined and back extracted with DCM(2×100 mL). The organic phases were combined and dried over MgSO₄,filtered and concentrated to an oil that was re-dissolved in ethanol(100 mL). Potassium fluoride (3.6 g, 63 mmol) was added to the solution,which was then brought to reflux for 1 hr. The mixture was cooled,concentrated in vacuo, re-dissolved in DCM (200 mL), filtered andconcentrated to an oil/crystal mixture. The mixture was re-dissolved ina minimum of DCM and loaded onto a silica gel column which waspre-equilibrated and eluted with 25% Acetone/Hexanes with 3% TEA toafford 2.70 g (74%) of a colorless wax.

Linoleyl Mesylate (7)

Linoleyl alcohol (10.0 g, 37.5 mmol) was weighed into a 500 mL roundbottomed flask with a stir bar. The flask was sealed, flushed withargon, charged with DCM (100 mL) and triethylamine (7.84 mL, 1.5 equiv.)and cooled to 0° C. Methanesulfonyl chloride (4.35 mL), 1.5 equiv.) wasmeasured in a PP syringe and added slowly to the stirring reactionmixture. TLC analysis (7.5% EtOAc/Hexanes) showed the reaction wascomplete within 1 hr. The reaction was diluted with DCM (100 mL) andwashed with saturated bicarbonate solution (2×200 mL). The organic phasewas dried over MgSO₄, filtered and concentrated to give 12.53 g (97%) ofcolorless oil that was used without further purification.

3-Dimethylamino-2-(cholest-5-en-3β-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(8a) (CLinDMA)

3-Dimethylamino-2-(Cholest-5-en-3β-oxybutan-4-oxy)-1-propanol (2.6 g,4.6 mmol) was weighed into a 200 mL round bottomed flask andco-evaporated with anhydrous toluene 2×20 mL). A stir bar was added tothe flask, which was then sealed, flushed with argon and charged withanhydrous toluene (100 mL). Sodium hydride (0.7 g, 6 equiv) was added atonce and the mixture was stirred, under argon, for 20 minutes. Linoleylmesylate (4.6 g, 2.3 equiv.) was measured in a PP syringe and addedslowly to the reaction mixture. The flask was fitted with a refluxcondenser and the apparatus was flushed with argon. The reaction mixturewas heated in an oil bath and allowed to stir at reflux overnight. Thereaction mixture was then cooled to room temperature in a water bath andethanol was added dropwise until gas evolution ceased. The reactionmixture was diluted with ethyl acetate (300 mL) and washed with aqueous10% sodium carbonate (2×200 mL). The aqueous phases were combined andback extracted with ethyl acetate (2×100 mL). The organic phases werecombined, dried over MgSO₄, filtered and concentrated. The resultant oilwas purified via column chromatography (10% EtOAc/Hexanes, 3% TEA) toafford 3.0 g (81%) of a colorless oil.

3-Dimethylamino-2-(cholest-5-en-3β-oxypent-3-oxa-an-5-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(DEGCLinDMA) (8b)

This compound was prepared similarly to3-Dimethylamino-2-(Cholest-5-en-3β-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane.3-Dimethylamino-2-(Cholest-5-en-3β-oxypent-3-oxa-an-5-oxy)-1-propanol(0.73 g, 1.3 mmol) was weighed into a 100 mL round bottomed flask andco-evaporated with anhydrous toluene. A stir bar was added to the flask,which was then sealed, flushed with argon and charged with anhydroustoluene. Sodium hydride (121 mg, 4 equiv.) was added at once and themixture was stirred, under argon, for 20 minutes. Linoleyl mesylate(0.873 g, 2 equiv.) was measured in a PP syringe and added slowly to thereaction mixture. The flask was fitted with a reflux condenser and theapparatus was flushed with argon. The reaction mixture was heated in anoil bath and allowed to stir at reflux overnight. The reaction mixturewas then cooled to room temperature in a water bath and ethanol wasadded dropwise until gas evolution ceased. The reaction mixture wasdiluted with ethyl acetate (150 mL) and washed with aqueous 10% sodiumcarbonate (2×100 mL). The aqueous phases were combined and backextracted with ethyl acetate (2×50 mL). The organic phases werecombined, dried over Na₂SO₄, filtered and concentrated. The resultantoil was purified via column chromatography (15% EtOAc/Hexanes, 3% TEA)to afford 0.70 g (67%) of colorless oil.

Alternative Route for Synthesis of CLinDMA (FIG. 29B)1-(t-Butyldimethylsilyloxy)-3-dimethylamino-2-propanol (6)

3-Dimethylamino-1,2-propanediol (5), (50.1 g, 420.4 mmol) was weighedinto a 2 L round bottomed flask with a stir bar. The flask was sealed,flushed with argon, charged with N,N-dimethylformamide (750 mL) andN,N-diisopropylethylamine (111 mL, 630.7 mmol) and cooled to 0° C.t-Butyldimethylchlorosilane (67.0 g, 1.05 equiv.) was weighed into a 500mL round bottomed flask, sealed and then dissolved inN,N-dimethylformamide (250 mL). The t-butyldimethylchlorosilane solutionwas transferred to a pressure equalizing dropping funnel and added tothe stirring reaction mixture slowly over 20 minutes. The reaction wasallowed to come to room temperature while stirring over 3 hours. Thereaction was concentrated in vacuo. Saturated bicarbonate (1500 mL) wasadded to the residue and the mixture transferred to a 4 L separatoryfunnel. The aqueous phase was extracted with ethyl acetate (3×500 mL).The organic phases were combined, dried over MgSO₄, filtered,concentrated and dried under high vacuum to afford 97.81 g (99.7%) ofclear, colorless oil that was used without further purification.

3-Dimethylamino-2-(cholest-5-en-3β-oxybutan-4-oxy)-1-propanol (7)

1-(t-Butyldimethylsilyloxy)-3-Dimethylamino-2-propanol (6) (25.52 g,109.3 mmol) was weighed into a two-necked 1 L round bottomed flaskcontaining a stir bar. The flask was fitted with a reflux condenser anda ground glass stopper, flushed with argon and charged with toluene (250mL). Sodium hydride (10.50 g, 4 equiv.) was added at once and themixture was stirred at room temperature for 20 minutes.Cholest-5-en-3β-oxybutan-4-mesylate (4, prepared as described above) wasdissolved in anhydrous toluene (100 mL) and added to the reactionmixture at once. An additional wash of toluene (30 mL) was used tofacilitate the complete transfer of residual mesylate to the reactionmixture. The flask was subjected to a continuous argon stream and thereaction was heated to reflux for 8 hrs. The reaction mixture was cooledto 0° C. in a water bath, diluted with ethyl acetate (350 mL) andethanol was added dropwise until gas evolution ceased. The reactionmixture was diluted with more ethyl acetate (350 mL) and washed withaqueous 10% sodium carbonate (2×1 L). The aqueous phases were combinedand back extracted with ethyl acetate (2×500 mL). The organic phaseswere combined, dried over MgSO₄, filtered and concentrated to an oil ina 2 L round bottomed flask.

The flask was fitted with a stir bar and the residue dissolved in amixture of dioxane (300 mL), ethanol (200 mL) and water (6 mL).Concentrated hydrochloric acid (11.3 mL, 139.2 mmoL) was added to thesolution which was then stirred for 2 hours at room temperature. 10%Sodium carbonate solution (2 L) was added to the reaction mixture in a 4L separatory funnel. The aqueous phase was extracted with ethyl acetate(3×750 mL). The organic phases were combined and dried over MgSO₄,filtered and concentrated to an oil. Purification of the oil wasperformed on a 4.5″ silica gel column pre-equilibrated with 3% TEA inhexanes. Elution was performed with 1 L of hexanes followed by 3 L of25% EtOAc/Hexanes with 3% TEA to afford 28.01 g (50.0%) of a colorlesswax.

Linoleyl Mesylate (8)

Linoleyl alcohol (10.0 g, 37.5 mmol) was weighed into a 500 mL roundbottomed flask with a stir bar. The flask was sealed, flushed withargon, charged with DCM (100 mL) and triethylamine (7.84 mL, 1.5 equiv.)and cooled to 0° C. Methanesulfonyl chloride (4.35 mL), 1.5 equiv.) wasmeasured in a PP syringe and added slowly to the stirring reactionmixture. TLC analysis (7.5% EtOAc/Hexanes) showed the reaction wascomplete within 1 hr. The reaction was diluted with DCM (100 mL) andwashed with saturated bicarbonate solution (2×200 mL). The organic phasewas dried over MgSO₄, filtered and concentrated to give 12.53 g (97%) ofcolorless oil that was used without further purification.

3-Dimethylamino-2-(cholest-5-en-3β-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA) (9)

3-Dimethylamino-2-(Cholest-5-en-3β-oxybutan-4-oxy)-1-propanol (7) (2.6g, 4.6 mmol) was weighed into a 200 mL round bottomed flask andco-evaporated with anhydrous toluene 2×20 mL). A stir bar was added tothe flask, which was then sealed, flushed with argon and charged withanhydrous toluene (100 mL). Sodium hydride (0.7 g, 6 equiv) was added atonce and the mixture was stirred, under argon, for 20 minutes. Linoleylmesylate (4.6 g, 2.3 equiv.) was measured in a PP syringe and addedslowly to the reaction mixture. The flask was fitted with a refluxcondenser and the apparatus was flushed with argon. The reaction mixturewas heated in an oil bath and allowed to stir at reflux overnight. Thereaction mixture was then cooled to room temperature in a water bath andethanol was added dropwise until gas evolution ceased. The reactionmixture was diluted with ethyl acetate (300 mL) and washed with aqueous10% sodium carbonate (2×200 mL). The aqueous phases were combined andback extracted with ethyl acetate (2×100 mL). The organic phases werecombined, dried over MgSO₄, filtered and concentrated. The resultant oilwas purified via column chromatography (10% EtOAc/Hexanes, 3% TEA) toafford 3.0 g (81%) of a colorless oil.

Example 17 Preparation of Aromatic Lipids of the Invention (see FIG.29C) Dioleyloxybenzaldehyde, 3a

3,4-Dihydroxybenzaldehyde (2.76 g, 20.0 mmol) was weighed into a 200 mLround bottomed flask with a stir bar. The flask was charged with diglyme(100 mL), septum sealed and flushed with argon. Cesium carbonate (19.5g, 60.0 mmol) was added to the solution slowly in portions. Oleylmesylate (15.2 g, 44.0 mmol) was added via syringe. The reaction mixturewas heated to 100° C. under slight positive pressure of argon. Thereaction mixture was cooled to room temperature and filtered. The solidswere washed with 1,2-dichloroethane. The combined filtrate and washeswere concentrated and then dried under high vacuum at 65° C. to removeresidual diglyme. The resultant yellow oil was purified via flashchromatography (5% ethyl acetate in hexanes) to afford 11.4 g (89%) of ayellow oil that turned to yellow wax upon standing at room temperature.

Dilinoleylbenzaldehyde, 3b

3,4-Dihydroxybenzaldehyde (2.76 g, 20.0 mmol) was weighed into a 200 mLround bottomed flask with a stir bar. The flask was charged with diglyme(100 mL), septum sealed and flushed with argon. Cesium carbonate (19.5g, 60.0 mmol) was added to the solution slowly in portions. Linoleylmesylate (15.2 g, 44.0 mmol) was added via syringe. The reaction mixturewas heated to 100° C. under slight positive pressure of argon. Thereaction mixture was cooled to room temperature and filtered. The solidswere washed with 1,2-dichloroethane. The combined filtrate and washeswere concentrated and then dried under high vacuum at 65° C. to removeresidual diglyme. The resultant yellow oil was purified via flashchromatography (5% ethyl acetate in hexanes) to afford 110.9 g (94%) ofa brown oil.

N,N-Dimethyl-3,4-dioleyloxybenzylamine, 4a

To a solution of triethylamineamine (2.0 mL, 14 mmol) in ethanol (20 mL)was added dimethylamine hydrochloride (1.63 g, 20 mmol), titaniumtetraisopropoxide (5.96 mL, 20 mmol) and 3,4-dioleyloxybenzaldehyde(6.39 g, 10 mmol). The mixture was allowed to stir under argon for 10 hat room temperature. Sodium borohydride (0.57 g, 15 mmol) was added tothe reaction mixture which was then allowed to stir at room temperatureovernight. Concentrated aqueous ammonia (4 mL) was added slowly to thereaction mixture. The reaction mixture was filtered and the solidswashed with dichloromethane. The filtrate was dried over K₂CO₃, filteredand concentrated. The resultant oil was purified via flashchromatography (2-10% acetone in dichloromethane, 0.5% TEA gradient) toafford 5.81 g (87-+%) of a yellow oil.

N,N-Dimethyl-3,4-dilinoleyloxybenzylamine, 4b

To a solution of triethylamineamine (2.0 mL, 14 mmol) in ethanol (20 mL)was added dimethylamine hydrochloride (1.63 g, 20 mmol), titaniumtetraisopropoxide (5.96 mL, 20 mmol) and 3,4-dilinoleyloxybenzaldehyde(6.35 g, 10 mmol). The mixture was allowed to stir under argon for 10 hat room temperature. Sodium borohydride (0.57 g, 15 mmol) was added tothe reaction mixture which was then allowed to stir at room temperatureovernight. 6N Aqueous ammonia (30 mL), was added slowly to the reactionmixture followed by dichloromethane. The reaction mixture was filtered.The filtrate was dried over K₂CO₃, filtered and concentrated. Theresultant oil was purified via flash chromatography (2-10% acetone indichloromethane, 0.5% TEA gradient) to afford 4.94 g (74%) of a yellowoil.

Example 18 Preparation of PEG-Conjugates of the Invention (see FIGS. 30Aand 30B) PEG-DMB (FIG. 30A)1-[8′-(Cholest-5-en-3β-oxy)carboxamido-3′,6′-dioxaoctanyl]carbamoyl-methyl-poly(ethyleneglycol) (PEG-cholesterol)

To a 200-mL round-bottom flask charged with a solution of 2.0 g (0.89mmol) of 1-[8′-amino-3′,6′-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethyleneglycol), 22 mg (0.18 mmol) of 4-dimethylaminopyridine, and 0.93 mL (5.3mmol) of diisopropylethylamine in 20 mL of anhydrous THF, was added withstirring a solution of 1.20 g (2.67 mmol) of cholesterol chloroformatein 20 mL of anhydrous THF. The resulting reaction mixture was heated togentle reflux overnight. After cooled, the solvents were removed byrotary evaporation, and the resulting residue was applied onto a silicagel column for purification (methanol/dichloromethane 5:95 to 10:90).The chromatography yielded 2.43 g (91%) of white solid product.

3,4-Ditetradecoxylbenzyl-ω-methyl-poly(ethylene glycol) ether (PEG-DMB)

To a 100-mL round-bottom flask charged with a solution of 2.67 g (5.00mmol) of ditetradecoxylbenzyl alcohol in 20 mL of 1,4-dioxane, was added20 mL of 4.0 M HCl solution in 1,4-dioxane. The flask was then equippedwith a refluxing condenser, which was connected to a sodium bicarbonatesolution to absorb any evolved hydrogen chloride gas. After the reactionmixture was heated to 80 for 6 h, thin layer chromatography(dichloromethane as developing solvent) indicated the completion of thereaction. The solvent and the excessive reagent were completely removedunder vacuum by rotary evaporation to afford 2.69 g (97%) of gray solid3,4-ditetradecoxylbenzyl chloride. This crude material was employeddirectly for the next step reaction without further purification.

Poly(ethylene glycol) methyl ether (2.00 g, 1.00 mmol) was dried byco-evaporating with toluene (2×20 mL) under vacuum. The PEG utilized isPEG2000, a polydispersion which can typically vary from ˜1500 to ˜3000Da (i.e., where PEG(n) is about 33 to about 67, or on average ˜45). To asolution of the dried poly(ethylene glycol) in 30 mL of anhydroustoluene, was added with stirring 0.17 g (7.2 mmol) of sodium hydride inportions. Gas evolvement took place instantly. The resulting mixturecontinued to be stirred at 60 for 2 h to ensure the complete formationof oxide. A solution of 0.668 g (1.20 mmol) 3,4-ditetradecoxylbenzylchloride in 10 mL of anhydrous toluene was then introduced dropwise tothe above mixture. The reaction mixture was allowed to stir at 80overnight. After cooled, the reaction was quenched by the addition of 10mL of saturated ammonium chloride solution. The resulting mixture wasthen taken into 300 mL of dichloromethane, washed with saturatedammonium chloride (3×100 mL), dried over anhydrous sodium sulfate, andevaporated to dryness. The residue was purified by flash chromatography(methanol/dichloromethane 2:98 to 5:95) to furnish 1.24 g (49%) of graysolid of the desired product.

PEG-DMG (FIG. 30B)

1,2-dimyristoyl-sn-glycerol (DMG-OH) (1) (10.0 g) and1,1′-Carbonyldiimidazole (CDI) (2) (3.32 g, 1.05 eq) were added to a 250mL dry round bottom flask equipped with magnetic stir bar and rubbersepta under argon. The flask was charged with 50 mL anhydrous THF andthe resulting mixture stirred for 6 hours at room temperature. The stirbar was removed and the reaction mixture transferred to a 1 L separatoryfunnel with 350 mL ethyl acetate. The reaction mixture was washed with200 mL deionized water. The aqueous phase was removed and the washrepeated 2×, the organic phase was collected and dried over 10 gmagnesium sulphate with stirring. Filtration over sintered glassfollowed by evaporation in vacuo provided1,2-Dimyristoyl-3-propanoxy-carboximidazole (DMG-CDI) (3); 11.68 g, 99%.To a mixture of Methoxy-PEG-NH2 2K (PEG-amine) (4) (2.36 g);1,2-Dimyristoyl-3-propanoxy-carboximidazole (DMG-CDI) (3) (1.91 g, 3.0eq); and 4-(N,N-Dimethylamino)pyridine (DMAP) (0.025 g, 0.2 eq) in a 200mL round bottomed flask equipped with stir bar and rubber septum underargon was added THF (20 mL) and Diisopropylethylamine (DiPEA) (1.10 mL,6.0 eq). The PEG utilized is PEG2000, a polydispersion which cantypically vary from ˜1500 to ˜3000 Da (i.e., where PEG(n) is about 33 toabout 67, or on average ˜45). The solution was brought to reflux andstirred for 17 hours after which the reaction mixture was cooled to roomtemperature, the stir bar removed, and the reaction concentrated invacuo to provide crude1-[8′-(1,2-Dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-co-methyl-poly(ethyleneglycol) (PEG-DMG) (5), 4.31 g.

Example 19 Preparation of Nanoparticle Encapsulated siNA/CarrierFormulations General LNP Preparation

siNA nanoparticle solutions were prepared by dissolving siNAs and/orcarrier molecules in 25 mM citrate buffer (pH 4.0) at a concentration of0.9 mg/mL. Lipid solutions were prepared by dissolving a mixture ofcationic lipid (e.g., CLinDMA or DOBMA, see structures and ratios forFormulations in Table IV), DSPC, Cholesterol, and PEG-DMG (ratios shownin Table IV) in absolute ethanol at a concentration of about 15 mg/mL.The nitrogen to phosphate ratio was approximate to 3:1.

Equal volume of siNA/carrier and lipid solutions was delivered with twoFPLC pumps at the same flow rates to a mixing T connector. A backpressure valve was used to adjust to the desired particle size. Theresulting milky mixture was collected in a sterile glass bottle. Thismixture was then diluted slowly with an equal volume of citrate buffer,and filtered through an ion-exchange membrane to remove any freesiNA/carrier in the mixture. Ultra filtration against citrate buffer (pH4.0) was employed to remove ethanol (test stick from ALCO screen), andagainst PBS (pH 7.4) to exchange buffer. The final LNP was obtained byconcentrating to a desired volume and sterile filtered through a 0.2 μmfilter. The obtained LNPs were characterized in term of particle size,Zeta potential, alcohol content, total lipid content, nucleic acidencapsulated, and total nucleic acid concentration

LNP Manufacture Process

In a non-limiting example, a LNP-086 siNA/carrier formulation isprepared in bulk as follows. A process flow diagram for the process isshown in Table VIII which can be adapted for siNA/carrier cocktails (2siNA/carrier duplexes are shown) or for a single siNA/carrier duplex.The process consists of (1) preparing a lipid solution; (2) preparing asiNA/carrier solution; (3) mixing/particle formation; (4) Incubation;(5) Dilution; (6) Ultrafiltration and Concentration.

1. Preparation of Lipid Solution

Summary: To a 3-necked round bottom flask fitted with a condenser wasadded a mixture of CLinDMA, DSPC, Cholesterol, PEG-DMG, and Linoeylalcohol. Ethanol was then added. The suspension was stirred with a stirbar under Argon, and was heated at 30° C. using a heating mantlecontrolled with a process controller. After the suspension became clear,the solution was allowed to cool to room temperature.

Detailed Procedure for Formulating 8 L Batch of LNP

-   -   1. Depyrogenate a 3-necked 2 L round bottom flask, a condenser,        measuring cylinders, and two 10 L conical glass vessels.    -   2. Warm the lipids to room temperature. Tare the weight of the        round bottom flask. Transfer the CLinDMA (50.44 g) with a        pipette using a pipette aid into the 3-necked round bottom        flask.    -   3. Weigh DSPC (43.32 g), Cholesterol (5.32 g) and PEG-DMG        (6.96 g) with a weighing paper sequentially into the round        bottom flask.    -   4. Linoleyl alcohol (2.64 g) was weighed in a separate glass        vial (depyrogenated). Tare the vial first, and then transfer the        compound with a pipette into the vial.    -   5. Take the total weight of the round bottom flask with the        lipids in, subtract the tare weight. The error was usually much        less than i 1.0%.    -   6. Transfer one-eighth of the ethanol (1 L) needed for the lipid        solution into the round bottom flask.    -   7. The round bottom flask placed in a heating mantle was        connected to a J-CHEM process controller. The lipid suspension        was stirred under Argon with a stir bar and a condenser on top.        A thermocouple probe was put into the suspension through one        neck of the round bottom flask with a sealed adapter.    -   8. The suspension was heated at 30° C. until it became clear.        The solution was allowed to cool to room temperature and        transferred to a conical glass vessel and sealed with a cap.        2. Preparation of siNA/Carrier Solution

Summary: The siNA/carrier solution can comprise a single siNA duplex andor carrier or can alternately comprise a cocktail of two or more siNAduplexes and/or carriers. In the case of a single siNA/carrier duplex,the siNA/carrier is dissolved in 25 mM citrate buffer (pH 4.0, 100 mM ofNaCl) to give a final concentration of 0.9 mg/mL. In the case of acocktail of two siNA/carrier molecules, the siNA/carrier solutions areprepared by dissolving each siNA/carrier molecule in 50% of the totalexpected volume of a 25 mM citrate buffer (pH 4.0, 100 mM of NaCl) togive a final concentration of 0.9 mg/mL. This procedure is repeated forthe other siNA/carrier molecule. The two 0.9 mg/mL siNA/carriersolutions are combined to give a 0.9 mg/mL solution at the total volumecontaining two siNA molecules.

Detailed Procedure for formulating 8 L Batch of LNP with siNA Cocktail

-   -   1. Weigh 3.6 g times the water correction factor (Approximately        1.2) of siNA-1 powder into a sterile container such as the        Corning storage bottle.    -   2. Transfer the siNA to a depyrogenated 5 L glass vessel. Rinse        the weighing container 3× with of citrate buffer (25 mM, pH 4.0,        and 100mM NaCl) placing the rinses into the 5 L vessel, QS with        citrate buffer to 4 L.    -   3. Determine the concentration of the siNA solution with UV        spectrometer. Generally, take 20 μL from the solution, dilute 50        times to 1000 μL, and record the UV reading at A260 nm after        blanking with citrate buffer. Make a parallel sample and        measure. If the readings for the two samples are consistent,        take an average and calculate the concentration based on the        extinction coefficients of the siNAs. If the final concentration        is out of the range of 0.90±0.01 mg/mL, adjust the concentration        by adding more siNA/carrier powder, or adding more citrate        buffer.    -   4. Repeat for siNA-2.    -   5. In a 101 depyrogenated 10 L glass vessel transfer 4 L of each        0.9 mg/mL siNA solution

Sterile Filtration.

The process describes the procedure to sterile filter the Lipid/Ethanolsolution. The purpose is to provide a sterile starting material for theencapsulation process. The filtration process was run at an 80 mL scalewith a membrane area of 20 cm². The flow rate is 280 mL/min. Thisprocess is scaleable by increasing the tubing diameter and thefiltration area.

1. Materials

a. Nalgene 50 Silicone Tubing PN 8060-0040 Autoclaved b. Master FlexPeristaltic Pump Model 7520-40   i. Master flex Pump Head Model 7518-00c. Pall Acropak 20 0.8/0.2 μm sterile filter. PN 12203 d. Depyrogenated10 L glass vessel e. Autoclaved lid for glass vessel.

2. Procedure.

-   -   a. Place tubing into pump head. Set pump to 50% total pump speed        and measure flow for 1 minute with a graduated cylinder    -   b. Adjust pump setting and measure flow to 280 mL/min.    -   c. Set up Tubing with filter attach securely with a clamp.    -   d. Set up pump and place tubing into pump head.    -   e. Place the feed end of the tubing into the material to be        filtered.    -   f. Place the filtrate side of filter with filling bell into        depyrogenated glass vessel.    -   g. Pump material through filter until all material is filtered.

AKTA Pump Setup

1. Materials

a. AKTA P900 Pump b. Teflon tubing 2 mm ID × 3 mm OD 2 each × 20.5 cmUpchurch PN 1677 c. Teflon tubing 1 mm ID × 3 mm OD 6.5 cm Upchurch PN1675 d. Peek Tee 1 mm ID 1 each Upchurch PN P-714 e. ¼-28F to 10-32M 2each Upchurch PN P-652

-   -   f. ETFE Ferrule for 3.0 mm OD tubing 6 each Upchurch PN P-343×    -   g. Flangless Nut 6 each Upchurch PN P-345×    -   h. ETFE cap for 1/4-28 flat bottom fitting 1 each Upchurch PN        P-755    -   i. Argon Compressed gas    -   j. Regulator 0-60 psi    -   k. Teflon tubing    -   l. Peek Y fitting    -   m. Depyrogenated glassware conical base.2/pump    -   n. Autoclaved lids.    -   o. Pressure lids

2. Pump Setup

-   -   a. Turn pump on    -   b. Allow pump to perform self test    -   c. Make certain that there are no caps or pressure regulators        attached to tubing (This will cause the pumps to over pressure.)    -   d. Press “OK” to synchronize pumps    -   e. Turn knob 4 clicks clockwise to “Setup”—press “OK”    -   f. Turn knob 5 clicks clockwise to “Setup Gradient Mode”—press        “OK”    -   g. Turn knob 1 click clockwise to “D”—press “OK”    -   h. Press “Esc” twice

3. Pump Sanitization.

-   -   a. Place 1000 mL of 1 N NaOH into a 1 L glass vessel    -   b. Attach to pump with a pressure lid    -   c. Place 1000 mL of 70% Ethanol into a 1 L glass vessel    -   d. Attach to pump with a pressure lid.    -   e. Place a 2000 mL glass vessel below pump outlet.    -   f. Turn knob 1 click clockwise to “Set Flow Rate”—press “OK”    -   g. Turn knob clockwise to increase Flow Rate to 40 mL/min;        counter clockwise to decrease; press “OK” when desired Flow Rate        is set.    -   h. Set time for 40 minute.    -   i. Turn on argon gas at 10 psi.    -   j. Turn knob 2 clicks counter clockwise to “Run”—press “OK”, and        start timer.    -   k. Turn knob I click counter clockwise to “End Hold Pause”    -   l. When timer sounds Press “OK” on pump    -   m. Turn off gas    -   n. Store pump in sanitizing solutions until ready for use        (overnight?)

4. Pump Flow Check

-   -   a. Place 200 mL of Ethanol into a depyrogenated 500 mL glass        bottle.    -   b. Attach to pump with a pressure cap.    -   c. Place 200 mL of Sterile Citrate buffer into a 500 mL        depyrogenated glass bottle.    -   d. Attach to pump with a pressure cap.    -   e. Place a 100 mL graduated cylinder below pump outlet.    -   f. Turn knob 1 click clockwise to “Set Flow Rate”—press “OK”    -   g. Turn knob clockwise to increase Flow Rate to 40 mL/min;        counter clockwise to decrease; press “OK” when desired Flow Rate        is set.    -   h. Set time for 1 minute.    -   i. Turn on argon gas at 10 psi.    -   j. Turn knob 2 clicks counter clockwise to “Run”—press “OK”, and        start timer.    -   k. Turn knob I click counter clockwise to “End Hold Pause”    -   l. When timer sounds Press “OK” on pump    -   m. Turn off gas    -   n. Verify that 40 mL of the ethanol/citrate solution was        delivered.

3. Particle Formation—Mixing Step

-   -   o. Attach the sterile Lipid/Ethanol solution to the AKTA pump.

p. Attach the sterile siNA/carrier or siNA/carrier cocktail/Citratebuffer solution to the AKTA pump.

-   -   q. Attach depyrogenated received vessel (2× batch size) with lid    -   r. Set time for calculated mixing time.    -   s. Turn on Argon gas and maintain pressure between 5 to 10 psi.    -   t. Turn knob 2 clicks counter clockwise to “Run”—press “OK”, and        start timer.    -   u. Turn knob 1 click counter clockwise to “End Hold Pause”    -   v. When timer sounds Press “OK” on pump    -   w. Turn off gas

4. Incubation

-   -   The solution is held after mixing for a 22±2 hour incubation.        The incubation is at room temperature (20-25° C.) and the        in-process solution is protected from light.

5. Dilution.

-   -   The lipid siNA solution is diluted with an equal volume of        Citrate buffer. The solution is diluted with a dual head        peristaltic pump, set up with equal lengths of tubing and a Tee        connection. The flow rate is 360 mL/minute.

1. Materials

h. Nalgene 50 Silicone Tubing PN 8060-0040 Autoclaved i. Tee ¼′ ID j.Master Flex Peristaltic Pump Model 7520-40 i. Master flex Pump HeadModel 7518-00 ii. Master flex Pump Head Model 7518-00 k. Depyrogenated 2× 20 L glass vessel l. Autoclaved lids for glass vessels.

2. Procedure.

-   -   a. Attach two equal lengths of tubing to the Tee connector. The        tubing should be approximately 1 meter in length. Attach a third        piece of tubing approximately 50 cm to the outlet end of the Tee        connector.    -   b. Place the tubing apparatus into the dual pump heads.    -   c. Place one feed end of the tubing apparatus into an Ethanol        solution. Place the other feed end into an equal volume of        Citrate buffer.    -   d. Set the pump speed control 50%. Set a time for 1 minute.    -   e. Place the outlet end of the tubing apparatus into a 500 mL        graduated cylinder.    -   f. Turn on the pump and start the timer.    -   g. When the timer sounds stop the pump and determine the        delivered volume.    -   h. Adjust the pump flow rate to 360 mL/minute.    -   i. Drain the tubing when the flow rate is set.    -   j. Place one feed end of the tubing apparatus into the        Lipid/siNA solution. Place the other feed end into an equal        volume of Citrate buffer (16 L).    -   k. Place the outlet end of the tubing apparatus into the first        of 2×20 L depyrogenated glass vessels.    -   l. Set a timer for 90 minutes and start the pump. Visually        monitor the dilution progress to ensure that the flow rates are        equal.    -   m. When the receiver vessel is at 16 liters change to the next        vessel and collect 16 L.    -   n. Stop the pump when all the material has been transferred.

6. Ultrafiltration and Concentration

-   -   Summary: The ultrafiltration process is a timed process and the        flow rates must be monitored carefully. The membrane area has        been determined based on the volume of the batch. This is a two        step process; the first is a concentration step taking the        diluted material from 32 liters to 3600 mLs and a concentration        of approximately 2 mg/mL. The concentration step is 4 hours±15        minutes. The second step is a diafiltration step exchanging the        ethanol citrate buffer to Phosphate buffered saline. The        diafiltration step is 3 hours and again the flow rates must be        carefully monitored. During this step the ethanol concentration        is monitored by head space GC. After 3 hours (20 diafiltration        volumes) a second concentration is undertaken to concentrate the        solution to approximately 6 mg/mL or a volume of 1.2 liters.        This material is collected into a depyrogenated glass vessel.        The system is rinsed with 400 mL of PBS at high flow rate and        the permeate line closed. This material is collected and added        to the first collection. The expected concentration at this        point is 4.5 mg/mL. The concentration and volume are determined.

1. Materials

x. Quatroflow pump y. Flexstand system with autoclaved 5 L reservoir. z.Ultrafiltration membrane GE PN UFP-100-C-35A aa. PBS 0.05 μm filtered100 L bb. 0.5 N Sodium Hydroxide. cc. WFI dd. Nalgene 50 Silicone TubingPN 8060-0040 Autoclaved ee. Master Flex Peristaltic Pump Model 7520-40  i. Master flex Pump Head Model 7518-00 ff. Permeate collection vessels100 L capacity gg. Graduated cylinders depyrogenated 2 L, 11, 500 mL.

2. Procedure

-   -   a. System preparation.        -   i. Install the membrane in the Flexstand holder, using the            appropriate size sanitary fittings for the membrane. Attach            the Flexstand to the quatroflow pump. Attach tubing to the            retentate and permeate connections and place these in a            suitable waste container.        -   ii. Determine the system hold up volume.            -   1. Place 1 liter of WFI in the reservoir.            -   2. Clamp the permeate line.            -   3. Start the Quatroflow pump and recirculate until no                bubbles are present in the retentate line. Stop pump            -   4. Mark the reservoir and record the reading for 1                liter.            -   5. Add 200 mL of WFI to the reservoir and mark the 1200                mL level.        -   iii. Add 3 liters of 0.5 N sodium hydroxide to the reservoir            and flush through the retentate to waste. Add 3 L of 0.5 N            sodium hydroxide to the reservoir recirculate the retentate            line and flush through the permeate to waste. Add a third 3            L of 0.5 N sodium hydroxide to the reservoir and recirculate            through the permeate line to the reservoir for 30 minutes.            Store the system in 0.5 N sodium hydroxide overnight prior            to use.        -   iv. Flush the sodium hydroxide to waste.        -   v. Add 3 L WFI to the reservoir and flush the retentate to            waste until the pH is neutral, replace the WFI as necessary.            Return the retentate line to the reservoir.        -   vi. Add 3 Liters of WFI and flush the permeate line to waste            until the pH is neutral, replacing the WFI as necessary.            Drain system.        -   vii. Add 3 Liters of Citrate buffer to the reservoir. Flush            through the permeate line until pH is <5. Add citrate buffer            as necessary.        -   viii. Drain system.    -   b. LNP Concentration        -   i. Place a suitable length on tubing into the peristaltic            pump head.        -   ii. Place the feed end into the diluted LNP solution; place            the other end into the reservoir.        -   iii. Pump the diluted LNP solution into the reservoir to the            4 liter mark.        -   iv. Place the permeate line into a clean waste container.        -   v. Start the quatroflow pump and adjust the pump speed so            the permeate flow rate is 300 mL/min.        -   vi. Adjust the peristaltic pump to 300 mL/min so the liquid            level is constant at 4 L in the reservoir.        -   vii. When all the diluted LNP solution has been transferred            to the reservoir stop the peristaltic pump.        -   viii. Concentrate the diluted LNP solution to 3600 mL in 240            minutes by adjusting the pump speed as necessary.        -   ix. Monitor the permeate flow rate, pump setting and feed            and retentate pressures.    -   c. LNP Diafiltration        -   i. Place the feed tubing of the peristaltic pump into a            container containing 72 L of PBS (0.05 μm filtered).        -   ii. Start the peristaltic pump and adjust the flow rate to            maintain a constant volume of 3600 mL in the reservoir.        -   iii. Increase the Quatroflow pump flow rate to 400 mL/min.        -   iv. Monitor the permeate flow rate, pump setting and feed            and retentate pressures.        -   v. Monitor the ethanol concentration by GC        -   vi. The LNP solution is diafiltered with PBS (20 volumes)            for 180 minutes.        -   vii. Stop the peristaltic pump. Remove tubing from            reservoir.    -   d. Final concentration        -   i. Concentrate the LNP solution to the 1.2 Liter mark.        -   ii. Collect the LNP solution into a depyrogenated 2 L            graduated cylinder.        -   iii. Add 400 mL of PBS to the reservoir.        -   iv. Start the pump and recirculate for 2 minutes.        -   v. Collect the rinse and add to the collected LNP solution            in the graduated cylinder.        -   vi. Record the volume of the LNP solution.        -   vii. Transfer to a 2 L depyrogenated glass vessel.        -   viii. Label and refrigerate.    -   e. Clean system        -   i. Add 1 L WFI to the reservoir        -   ii. Recirculate for 5 minutes with permeate closed.        -   iii. Drain system        -   iv. Add 2 L 0.5 N sodium hydroxide to the reservoir        -   v. Recirculate for 5 minutes.        -   vi. Drain system        -   vii. Add 2 L of 0.5 N sodium hydroxide to the reservoir.        -   viii. Recirculate for 5 minutes and stop pump.        -   ix. Neutralize system with WFI.        -   x. Drain system and discard membrane.

The obtained LNPs were characterized in term of particle size, Zetapotential, alcohol content, total lipid content, nucleic acidencapsulated, and total nucleic acid concentration.

Example 20 Improving Activity as a Mixture

To explore if the carrier effect allows for efficient RNAi for multiplesiRNAs in a mixture, siRNAs were used targeting 3 different genes (seeFIG. 54). The SSB 291, CRTC2:283 and IKK2 2389 siRNAs were dosed at 3mg/kg alone or as a mixture of all three along with 2.1 mg/kg carrierHCV316 at a total dose of 3 mg/kg. When the siRNAs were dosedindividually at 0.3 mg/kg, they showed moderate to no knockdown of theirintended target. On the other hand, when given as mixture, knockdownefficiency was improved significantly. For SSB291, the target knockdownimproved from 31% when given alone to 77% when given in a mixture. ForCRTC2:283, the target knockdown improved from 17% when given alone to41% when given in a mixture. For IKK2:2389, no target knockdown wasobserved when given alone but it improved to 48% when given in amixture. Thus, even though the concentration of each siRNA was 0.3mg/kg, when given alone or in a mixture, significant improvement inactivity was achieved by dosing them as a mixture. This allows targetingof multiple targets at lower doses to achieve additive or synergisticeffects.

Example 21 Use of Empty LNP for Beneficial Carrier Effect

To further understand the nature of carrier cargo, empty L201 wasprepared. The SSB291 L201 was injected at 1 mg/kg alone, or in thepresence of empty L201 as carrier at a total dose of 3 mg/kg. The totalliver RNA was isolated and analysed for SSB RNA. As shown in FIG. 55,SSB291 showed 54% knockdown of target RNA when dosed alone but whensupplied with empty LNP carrier, the knockdown efficiency improved to79%. The carrier empty LNP on its own showed no significant knockdown ofSSB target. This result shows that potentiation of RNAi activity can beachieved by empty LNP, “empty carrier”.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims. The present invention teaches oneskilled in the art to test various combinations and/or substitutions ofchemical modifications described herein toward generating nucleic acidconstructs with improved activity for mediating RNAi activity. Suchimproved activity can comprise improved stability, improvedbioavailability, and/or improved activation of cellular responsesmediating RNAi. Therefore, the specific embodiments described herein arenot limiting and one skilled in the art can readily appreciate thatspecific combinations of the modifications described herein can betested without undue experimentation toward identifying siNA moleculeswith improved RNAi activity.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments, optional features,modification and variation of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention asdefined by the description and the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

TABLE I Non-limiting examples of Stabilization Chemistries forchemically modified siNA constructs Chemistry pyrimidine Purine cap p =S Strand “Stab 00” Ribo Ribo TT at 3′-ends S/AS “Stab 1” Ribo Ribo — 5at 5′-end S/AS 1 at 3′-end “Stab 2” Ribo Ribo — All linkages Usually AS“Stab 3” 2′-fluoro Ribo — 4 at 5′-end Usually S 4 at 3′-end “Stab 4”2′-fluoro Ribo 5′ and 3′-ends — Usually S “Stab 5” 2′-fluoro Ribo — 1 at3′-end Usually AS “Stab 6” 2′-O-Methyl Ribo 5′ and 3′- — Usually S ends“Stab 7” 2′-fluoro 2′-deoxy 5′ and 3′- — Usually S ends “Stab 8”2′-fluoro 2′-O-Methyl — 1 at 3′-end S/AS “Stab 9” Ribo Ribo 5′ and 3′- —Usually S ends “Stab 10” Ribo Ribo — 1 at 3′-end Usually AS “Stab 11”2′-fluoro 2′-deoxy — 1 at 3′-end Usually AS “Stab 12” 2′-fluoro LNA 5′and 3′- Usually S ends “Stab 13” 2′-fluoro LNA 1 at 3′-end Usually AS“Stab 14” 2′-fluoro 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end “Stab15” 2′-deoxy 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end “Stab 16” Ribo2′-O- 5′ and 3′- Usually S Methyl ends “Stab 17” 2′-O-Methyl 2′-O- 5′and 3′- Usually S Methyl ends “Stab 18” 2′-fluoro 2′-O- 5′ and 3′-Usually S Methyl ends “Stab 19” 2′-fluoro 2′-O- 3′-end S/AS Methyl “Stab20” 2′-fluoro 2′-deoxy 3′-end Usually AS “Stab 21” 2′-fluoro Ribo 3′-endUsually AS “Stab 22” Ribo Ribo 3′-end Usually AS “Stab 23” 2′-fluoro*2′-deoxy* 5′ and 3′- Usually S ends “Stab 24” 2′-fluoro* 2′-O- — 1 at3′-end S/AS Methyl* “Stab 25” 2′-fluoro* 2′-O- — 1 at 3′-end S/ASMethyl* “Stab 26” 2′-fluoro* 2′-O- — S/AS Methyl* “Stab 27” 2′-fluoro*2′-O- 3′-end S/AS Methyl* “Stab 28” 2′-fluoro* 2′-O- 3′-end S/AS Methyl*“Stab 29” 2′-fluoro* 2′-O- 1 at 3′-end S/AS Methyl* “Stab 30” 2′-fluoro*2′-O- S/AS Methyl* “Stab 31” 2′-fluoro* 2′-O- 3′-end S/AS Methyl* “Stab32” 2′-fluoro 2′-O- S/AS Methyl “Stab 33” 2′-fluoro 2′-deoxy* 5′ and 3′-— Usually S ends “Stab 34” 2′-fluoro 2′-O- 5′ and 3′- Usually S Methyl*ends “Stab 35” 2′-fluoro** 2′-O- Usually AS Methyl** “Stab 36”2′-fluoro** 2′-O- Usually AS Methyl** “Stab 3F” 2′-OCF3 Ribo — 4 at5′-end Usually S 4 at 3′-end “Stab 4F” 2′-OCF3 Ribo 5′ and 3′- — UsuallyS ends “Stab 5F” 2′-OCF3 Ribo — 1 at 3′-end Usually AS “Stab 7F” 2′-OCF32′-deoxy 5′ and 3′- — Usually S ends “Stab 8F” 2′-OCF3 2′-O- — 1 at3′-end S/AS Methyl “Stab 11F” 2′-OCF3 2′-deoxy — 1 at 3′-end Usually AS“Stab 12F” 2′-OCF3 LNA 5′ and 3′- Usually S ends “Stab 13F” 2′-OCF3 LNA1 at 3′-end Usually AS “Stab 14F” 2′-OCF3 2′-deoxy 2 at 5′-end UsuallyAS 1 at 3′-end “Stab 15F” 2′-OCF3 2′-deoxy 2 at 5′-end Usually AS 1 at3′-end “Stab 18F” 2′-OCF3 2′-O- 5′ and 3′- Usually S Methyl ends “Stab19F” 2′-OCF3 2′-O- 3′-end S/AS Methyl “Stab 20F” 2′-OCF3 2′-deoxy 3′-endUsually AS “Stab 21F” 2′-OCF3 Ribo 3′-end Usually AS “Stab 23F” 2′-OCF3*2′-deoxy* 5′ and 3′- Usually S ends “Stab 24F” 2′-OCF3* 2′-O- — 1 at3′-end S/AS Methyl* “Stab 25F” 2′-OCF3* 2′-O- — 1 at 3′-end S/AS Methyl*“Stab 26F” 2′-OCF3* 2′-O- — S/AS Methyl* “Stab 27F” 2′-OCF3* 2′-O-3′-end S/AS Methyl* “Stab 28F” 2′-OCF3* 2′-O- 3′-end S/AS Methyl* “Stab29F” 2′-OCF3* 2′-O- 1 at 3′-end S/AS Methyl* “Stab 30F” 2′-OCF3* 2′-O-S/AS Methyl* “Stab 31F” 2′-OCF3* 2′-O- 3′-end S/AS Methyl* “Stab 32F”2′-OCF3 2′-O- S/AS Methyl “Stab 33F” 2′-OCF3 2′-deoxy* 5′ and 3′- —Usually S ends “Stab 34F” 2′-OCF3 2′-O- 5′ and 3′- Usually S Methyl*ends “Stab 35F” 2′-OCF3*† 2′-O- Usually AS Methyl*† “Stab 36F” 2′-OCF3*†2′-O- Usually AS Methyl*† CAP = any terminal cap. All Stab 00-34chemistries can comprise 3′-terminal thymidine (TT) residues All Stab00-34 chemistries typically comprise about 21 nucleotides, but can varyas described herein. All Stab 00-36 chemistries can also include asingle ribonucleotide in the sense or passenger strand at the 11^(th)base paired position of the double stranded nucleic acid duplex asdetermined from the 5′-end of the antisense or guide strand S = sensestrand AS = antisense strand *Stab 23 has a single ribonucleotideadjacent to 3′-CAP *Stab 24 and Stab 28 have a single ribonucleotide at5′-terminus *Stab 25, Stab 26, Stab 27, Stab 35 and Stab 36 have threeribonucleotides at 5′-terminus *Stab 29, Stab 30, Stab 31, Stab 33, andStab 34 any purine at first three nucleotide positions from 5′-terminusare ribonucleotides p = phosphorothioate linkage †Stab 35 has2′-O-methyl U at 3′-overhangs and three ribonucleotides at 5′-terminus†Stab 36 has 2′-O-methyl overhangs that are complementary to the targetsequence (naturually occurring overhangs) and three ribonucleotides at5′-terminus

TABLE II Wait Time* Wait Time* 2′-O- Wait Reagent Equivalents Amount DNAmethyl Time*RNA A. 2.5 μmol Synthesis Cycle ABI 394 InstrumentPhosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min S-Ethyl 23.8 238 μL45 sec 2.5 min 7.5 min Tetrazole Acetic 100 233 μL  5 sec  5 sec  5 secAnhydride N-Methyl 186 233 μL  5 sec  5 sec  5 sec Imidazole TCA 176 2.3 mL 21 sec 21 sec 21 sec Iodine 11.2  1.7 mL 45 sec 45 sec 45 secBeaucage 12.9 645 μL 100 sec  300 sec  300 sec  Acetonitrile NA 6.67 mLNA NA NA B. 0.2 μmol Synthesis Cycle ABI 394 Instrument Phosphoramidites15  31 μL 45 sec 233 sec  465 sec  S-Ethyl 38.7  31 μL 45 sec 233 min  465 sec  Tetrazole Acetic 655 124 μL  5 sec  5 sec  5 sec AnhydrideN-Methyl 1245 124 μL  5 sec  5 sec  5 sec Imidazole TCA 700 732 μL 10sec 10 sec 10 sec Iodine 20.6 244 μL 15 sec 15 sec 15 sec Beaucage 7.7232 μL 100 sec  300 sec  300 sec  Acetonitrile NA 2.64 mL NA NA NA C.0.2 μmol Synthesis Cycle 96 well Instrument Equivalents: Amount:DNA/2′-O- DNA/2′-O- Wait Time* Wait Time* Wait Time* Reagent methyl/Ribomethyl/Ribo DNA 2′-O-methyl Ribo Phosphoramidites 22/33/66 40/60/120 μL60 sec 180 sec 360 sec  S-Ethyl 70/105/210 40/60/120 μL 60 sec 180 min360 sec  Tetrazole Acetic 265/265/265 50/50/50 μL 10 sec  10 sec 10 secAnhydride N-Methyl 502/502/502 50/50/50 μL 10 sec  10 sec 10 secImidazole TCA 238/475/475 250/500/500 μL 15 sec  15 sec 15 sec Iodine6.8/6.8/6.8 80/80/80 μL 30 sec  30 sec 30 sec Beaucage 34/51/5180/120/120 100 sec  200 sec 200 sec  Acetonitrile NA 1150/1150/1150 μLNA NA NA Wait time does not include contact time during delivery. Tandemsynthesis utilizes double coupling of linker molecule

TABLE III Structure NAME Abbrev.

Cholesterol Chol

Cholest-5-en-3β-tosylate Chol-OTs

Cholest-5-en-3β-oxybutan- 4-ol Chol-OBu-OH

Cholest-5-en-3β-oxypent- 3-oxa-an-5-ol Chol-DEG-OH

Cholest-5-en-3β-oxybutan- 4-mesylate

Cholest-5-en-3β-oxypent-3- oxa-an-5-mesylate

3-Dimethylamino-1,2- propanediol

1-(4,4′-Dimethoxytrityloxy)-3- Dimethylamino-2-propanol

3-Dimethylamino-2-(Cholest- 5-en-3β-oxybutan-4-oxy)-1- propanol

3-Dimethylamino-2-(Cholest- 5-en-3β-oxypent-3-oxa-an-5- oxy)-1-propanol

cis,cis-9,12-octadecadiene-1- ol (linoleyl alcohol) Lin-OH

cis,cis-9,12-octadecadiene- mesylate (linoleyl mesylate) Lin-OMs

3-Dimethylamino-2-(Cholest- 5-en-3β-oxybutan-4-oxy)-1- (cis,cis-9,12-octadecadienoxy)propane CLinDMA

3-Dimethylamino-2-(Cholest- 5-en-3β-oxypent-3-oxa-an-5-oxy)-1-(cis,cis-9,12- octadecadienoxy)propane DEG-CLinDMA

Chol-oBu-Im

Chol-oBu-2MeIm CLinDMA structure

pCLinDMA structure

eCLinDMA structure

DEGCLinDMA structure

PEG-n-DMG structure

DMOBA structure

DMLBA structure

DOBA structure

DSPC structure

Cholesterol structure

2KPEG-Cholesterol structure

2KPEG-DMG structure

COIM STRUCTURE

5-CLIM AND 2-CLIM STRUCTURE

TABLE IV Lipid Nanoparticle (LNP) Formulations Formu- lation #Composition Molar Ratio L051 CLinDMA/DSPC/Chol/PEG-n-DMG 48/40/10/2 L053DMOBA/DSPC/Chol/PEG-n-DMG 30/20/48/2 L054 DMOBA/DSPC/Chol/PEG-n-DMG50/20/28/2 L069 CLinDMA/DSPC/Cholesterol/PEG- 48/40/10/2 CholesterolL073 pCLinDMA or CLin DMA/DMOBA/DSPC/ 25/25/20/28/2 Chol/PEG-n-DMG L077eCLinDMA/DSPC/Cholesterol/2KPEG- 48/40/10/2 Chol L080eCLinDMA/DSPC/Cholesterol/2KPEG- 48/40/10/2 DMG L082pCLinDMA/DSPC/Cholesterol/2KPEG- 48/40/10/2 DMG L083pCLinDMA/DSPC/Cholesterol/2KPEG- 48/40/10/2 Chol L086CLinDMA/DSPC/Cholesterol/2KPEG- 43/38/10/2/7 DMG/Linoleyl alcohol L061DMLBA/Cholesterol/2KPEG-DMG 52/45/3 L060 DMOBA/Cholesterol/2KPEG-DMG N/Pratio 52/45/3 of 5 L097 DMLBA/DSPC/Cholesterol/2KPEG-DMG 50/20/28 L098DMOBA/Cholesterol/2KPEG-DMG, N/P 52/45/3 ratio of 3 L099DMOBA/Cholesterol/2KPEG-DMG, N/P 52/45/3 ratio of 4 L100 DMOBA/DOBA/3%PEG-DMG, N/P ratio of 3 52/45/3 L101 DMOBA/Cholesterol/2KPEG-Cholesterol52/45/3 L102 DMOBA/Cholesterol/2KPEG-Cholesterol, 52/45/3 N/P ratio of 5L103 DMLBA/Cholesterol/2KPEG-Cholesterol 52/45/3 L104CLinDMA/DSPC/Cholesterol/2KPEG- 43/38/10/2/7 cholesterol/Linoleylalcohol L105 DMOBA/Cholesterol/2KPEG-Chol, N/P ratio 52/45/3 of 2 L106DMOBA/Cholesterol/2KPEG-Chol, N/P ratio 67/30/3 of 3 L107DMOBA/Cholesterol/2KPEG-Chol, N/P ratio 52/45/3 of 1.5 L108DMOBA/Cholesterol/2KPEG-Chol, N/P ratio 67/30/3 of 2 L109DMOBA/DSPC/Cholesterol/2KPEG-Chol, 50/20/28/2 N/P ratio of 2 L110DMOBA/Cholesterol/2KPEG-DMG, N/P 52/45/3 ratio of 1.5 L111DMOBA/Cholesterol/2KPEG-DMG, N/P 67/30/3 ratio of 1.5 L112DMLBA/Cholesterol/2KPEG-DMG, N/P ratio 52/45/3 of 1.5 L113DMLBA/Cholesterol/2KPEG-DMG, N/P ratio 67/30/3 of 1.5 L114DMOBA/Cholesterol/2KPEG-DMG, N/P 52/45/3 ratio of 2 L115DMOBA/Cholesterol/2KPEG-DMG, N/P 67/30/3 ratio of 2 L116DMLBA/Cholesterol/2KPEG-DMG, N/P ratio 52/45/3 of 2 L117DMLBA/Cholesterol/2KPEG-DMG, N/P ratio 52/45/3 of 2 L118LinCDMA/DSPC/Cholesterol/2KPEG- 43/38/10/2/7 DMG/Linoleyl alcohol, N/Pratio of 2.85 L121 2-CLIM/DSPC/Cholesterol/2KPEG-DMG/, 48/40/10/2 N/Pratio of 3 L122 2-CLIM/Cholesterol/2KPEG-DMG/, N/P 68/30/2 ratio of 3L123 CLinDMA/DSPC/Cholesterol/2KPEG- 43/38/10/3/7 DMG/Linoleyl alcohol,N/P ratio of 2.85 L124 CLinDMA/DSPC/Cholesterol/2KPEG- 43/36/10/4/7DMG/Linoleyl alcohol, N/P ratio of 2.85 L130CLinDMA/DOPC/Chol/PEG-n-DMG, 48/39/10/3 N/P ratio of 3 L131DMLBA/Cholesterol/2KPEG-DMG, N/P ratio 52/43/5 of 3 L132DMOBA/Cholesterol/2KPEG-DMG, N/P ratio 52/43/5 of 3 L133CLinDMA/DOPC/Chol/PEG-n-DMG, 48/40/10/2 N/P ratio of 3 L134CLinDMA/DOPC/Chol/PEG-n-DMG, 48/37/10/5 N/P ratio of 3 L149COIM/DSPC/Cholesterol/2KPEG-DMG/, N/P 48/40/10/2 ratio of 3 L155CLinDMA/DOPC/Cholesterol/2KPEG- 43/38/10/2/7 DMG/Linoleyl alcohol, N/Pratio of 2.85 L156 CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 45/43/10/2 N/Pratio of 2.85 L162 CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 45/43/10/2 N/Pratio of 2.5 L163 CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 45/43/10/2 N/Pratio of 2 L164 CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 45/43/10/2 N/P ratioof 2.25 L165 CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 40/43/15/2 N/P ratio of2.25 L166 CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 40/43/15/2 N/P ratio of2.5 L167 CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 40/43/15/2 N/P ratio of 2L174 CLinDMA/DSPC/DOPC/Cholesterol/2KPEG- 43/9/27/10/4/7 DMG/Linoleylalcohol, N/P ratio of 2.85 L175 CLinDMA/DSPC/DOPC/Cholesterol/2KPEG-43/27/9/10/4/7 DMG/Linoleyl alcohol, N/P ratio of 2.85 L176CLinDMA/DOPC/Cholesterol/2KPEG- 43/38/10/4/7 DMG/Linoleyl alcohol, N/Pratio of 2.85 L180 CLinDMA/DOPC/Cholesterol/2KPEG- 43/38/10/4/7DMG/Linoleyl alcohol, N/P ratio of 2.25 L181CLinDMA/DOPC/Cholesterol/2KPEG- 43/38/10/4/7 DMG/Linoleyl alcohol, N/Pratio of 2 L182 CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 45/41/10/4 N/P ratioof 2.25 L197 CODMA/DOPC/Cholesterol/2KPEG-DMG, 43/36/10/4/7 N/P ratio of2.85 L198 CLinDMA/DOPC/Cholesterol/2KPEG- 43/34/10/4/2/7DMG/2KPEG-DSG/Linoleyl alcohol, N/P ratio of 2.85 L199CLinDMA/DOPC/Cholesterol/2KPEG- 43/34/10/6/7 DMG/Linoleyl alcohol, N/Pratio of 2.85 L200 CLinDMA/Cholesterol/2KPEG-DMG, N/P 50/46/4 ratio of3.0 L201 CLinDMA/Cholesterol/2KPEG-DMG, N/P 50/44/6 ratio of 3.0 L206CLinDMA/Cholesterol/2KPEG-DMG, N/P 40/56/4 ratio of 3.0 L207CLinDMA/Cholesterol/2KPEG-DMG, N/P 60/36/4 ratio of 3.0 L208CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 40/10/46/4 N/P ratio of 3.0 L209CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 60/10/26/4 N/P ratio of 3.0 L210CLinDMA/Chol-oBu-Im/PEG-DMG 50/46/4 L211 CLinDMA/Chol-oBu-Im/PEG-DMG50/44/6 L212 CLinDMA/Chol-oBu-2MeIm/PEG-DMG 50/44/6 L216CLinDMA/Chol-oBu-Im/PEG-DMG 68/26/6 L217 CLinDMA/Chol-oBu-2MeIm/PEG-DMG68/26/6 L219 CLinDMA/Chol/Chol-oBu-2MeIm/PEG- 50/18/26/6 DMG L220CLinDMA/Chol/Chol-oBu-2MeIm/PEG- 40/28/26/6 DMG L222pCLinDMA/Chol/PEG-DMG 50/44/6 L223 eCLinDMA/Chol/PEG-DMG 50/44/6 L224buteneCLinDMA/Chol/PEG-DMG 50/44/6 L237 COleyl-2MeIm/Chol/PEG-DMG50/44/6 L238 Clin-Im/Chol/PEG-DMG 50/44/6 L239 Coleyl-Im/Chol/PEG-DMG50/44/6 L240 CLin-2MeIm/Chol/PEG-DMG 50/44/6 B083 DMOBA/Chol/PEG-DMG50/48/2 N/P ratio = Nitrogen:Phosphorous ratio between cationic lipidand nucleic acid The 2KPEG utilized is PEG2000, a polydispersion whichcan typically vary from ~1500 to ~3000 Da (i.e., where PEG(n) is about33 to about 67, or on average ~45).

TABLE V Description of pattern Pattern # Score G or C at position 1 1 5A or U at position 19 2 10 A/U rich between position 15-19 3 10 Stringof 4 Gs or 4 Cs (not 4 −100 preferred) G/C rich between position 1-5 510 A or U at position 18 6 5 A or U at position 10 7 10 G at position 13(not preferred) 8 −3 A at position 13 9 3 G at position 9 (notpreferred) 10 −3 A at position 9 11 3 A or U at position 14 12 10 Sirnaalgorithm describing patterns with their relative score for predictinghyper-active siNAs. All the positions given are for the sense strand of1

TABLE VI Manufacturing Flow Diagram

TABLE VII Analtical Methods for siNA characterization Acceptance TestName Test Method Criteria Results Identity Tests ID HPLC Retention timeof Oligonucleotide: sample and Main Peak standard main peaks comparefavorably Molecular Weight MS MW = N ± 3 amu amu (sodium free) MeltingUV monitored ° C. Temperature Assay Tests Oligonucleotide UV NLT 800μg/mg μg/mg Content Purity Tests Oligonucleotide HPLC NLT 80.0% Main %Duplex: Main Peak Peak Oligonucleotide: HPLC monitored % Total OtherRelated Substances Oligonucleotide HPLC Identify strand % Single Strandand quantity, monitored Oligonucleotide HPLC monitored % Duplex: MainPeak Other Tests Physical Visual White to pale Appearance yellow powderpH pH monitored Bacterial LAL <dose dependent> EU/mg Endotoxins AerobicMicrobiology NMT 500 CFU/gram Bioburden CFU/gram Residual GC NMT 410 ppmppm Acetonitrile Water Content Karl Fischer monitored % Metals ContentICP monitored Aluminum =      ppm Nickel =      ppm Chromium =      ppmMolybdenum =      ppm Copper =      ppm Iron =      ppm Magnesium =     ppm Ions Content AA and Ion monitored Sodium =      % ChromatographyChloride =      ppm Phosphate =      ppm

TABLE VIII LNP PROCESS FLOW CHART

*siNA 2 is optional, shown for input into LNP siNA cocktail formulation,additional siNA duplexes, e.g., siNA 3, siNA 4, siNA 5 etc. can be usedfor siNA cocktails

TABLE IX SEQ SEQ SEQ Sense ID Antisense ID ID Aliases sequence NO:sequence NO: Target seq NO: HBV:(263)21 BGGAcuucuc 12 AGAAAAuuGAG 16GGACUUC 20 siNA stab07/35 ucAAuuuucuT AGAAGuccUU UCUCAAU active TB UUUCUHCVa:(316)21 BccGGGAGG 13 GGUcuAcGAGAcc 17 CCGGGAG 21 siNA stab07/35ucucGuAGAc ucccGGUU GUCUCGU active cTTB AGACC HBV:(263)21 BucuuuuAAcu 14ccuGAAGAGAGu 18 GGACUUC 22 siNA stab07/35A cucuucAGGT uAAAAGAUU UCUCAAUinvert T UUUCU SSB:(291)21 BAcAAcAGAc 15 UUAcAuuAAAGu 19 ACAACAG 23 siNAstab07/35 uuuAAuGuA cuGuuGuUU ACUUUAA active ATTB UGUAA Uppercase= ribonucleotide u = 2′-deoxy-2′-fluoro uridine c = 2′-deoxy-2′-fluorocytidine g = 2′-deoxy-2′-fluoro guanosine a = 2′-deoxy-2′-fluoroadenosine T = thymidine B = inverted deoxy abasic s = phosphorothioatelinkage A = deoxy Adenosine G = deoxy Guanosine U = deoxy Uridine C= deoxy Cytidine G = 2′-O-methyl Guanosine A = 2′-O-methyl AdenosineC = 2′-O-methyl Cytidine U = 2′-O-methyl Uridine p = terminal phosphate

1. A composition comprising a first lipid nanoparticle (LNP) vehicle anda second lipid nanoparticle (LNP) vehicle each having size between about50 nm and about 500 nm, wherein: a) each vehicle comprises a cationiclipid, a neutral lipid, and a PEG-lipid; b) the first LNP vehiclefurther comprises one or more short interfering nucleic acid (siNA)molecules comprising a sense strand and a complementary antisensestrand, each strand having between 15 and 30 nucleotides in length,wherein the antisense strand comprises between 15 and 30 nucleotidesthat are complementary to a mammalian RNA sequence and the sense strandcomprises between 15 and 30 nucleotides of said mammalian RNA sequence;and c) the second LNP vehicle further comprises one or more carriermolecules comprising a nucleic acid sequence of at least 15 nucleotidesthat is not complementary to said mammalian RNA sequence; wherein thelipids of the first LNP vehicles are the same as or different from thesecond LNP vehicle.
 2. The composition of claim 1, wherein the lipidcomponents of each lipid nanoparticle vehicle are the same.
 3. Thecomposition of claim 1, wherein each lipid nanoparticle vehiclecomprises a different composition of lipid components.
 4. Thecomposition of claim 1, wherein the first and second lipid nanoparticlevehicles each comprise3-Dimethylamino-2-(Cholest-5-en-3β-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), distearoylphosphatidylcholine (DSPC), Cholesterol,and1-[8′-(1,2-Dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethyleneglycol) (PEG-DMG).
 5. The composition of claim 4, wherein each LNPvehicle further comprises Linoleyl alcohol.
 6. The composition of claim5, wherein said CLinDMA, DSPC, Cholesterol, PEG-DMG, and Linoleylalcohol have a molar ratio of about 43/36/10/4/7.
 7. (canceled) 8.(canceled)
 9. (canceled)
 10. (canceled)
 11. (canceled)
 12. (canceled)13. A composition comprising a lipid nanoparticle (LNP) vehicle havingsize between about 50 nm and about 500 nm, wherein: a) the lipidnanoparticle vehicle comprises a cationic lipid, a neutral lipid, and aPEG-lipid; b) the vehicle further comprises one or more shortinterfering nucleic acid (siNA) molecules comprising a sense strand anda complementary antisense strand, each strand having between 15 and 30nucleotides in length, wherein the antisense strand comprises between 15and 30 nucleotides that are complementary to a mammalian RNA sequence,and the sense strand comprises between 15 and 30 nucleotides of saidmammalian RNA sequence; c) the vehicle further comprises one or morecarrier molecules comprising a nucleic acid sequence of at least 15nucleotides that is not complementary to said mammalian RNA sequence.14. The composition of claim 13, wherein the lipid nanoparticle vehiclecomprises3-Dimethylamino-2-(Cholest-5-en-3β-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), distearoylphosphatidylcholine (DSPC), Cholesterol,and1-[8′-(1,2-Dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethyleneglycol) (PEG-DMG).
 15. The composition of claim 14, wherein the LNPvehicle further comprises Linoleyl alcohol.
 16. The composition of claim15, wherein said CLinDMA, DSPC, Cholesterol, PEG-DMG, and Linoleylalcohol have a molar ratio of about 43/36/10/4/7.
 17. (canceled) 18.(canceled)
 19. (canceled)
 20. (canceled)
 21. (canceled)
 22. (canceled)23. A composition comprising one or more carrier molecules and a lipidnanoparticle (LNP) vehicle having size between about 50 nm and about 500nm, wherein: a) the lipid nanoparticle vehicle comprises a cationiclipid, a neutral lipid, and a PEG-lipid; and b) the vehicle furthercomprises one or more short interfering nucleic acid (siNA) moleculescomprising a sense strand and a complementary antisense strand, eachstrand having between 15 and 30 nucleotides in length, wherein theantisense strand comprises between 15 and 30 nucleotides that arecomplementary to a mammalian RNA sequence, and the sense strandcomprises between 15 and 30 nucleotides of said mammalian RNA sequence.24. The composition of claim 23, wherein the lipid nanoparticle vehiclecomprises3-Dimethylamino-2-(Cholest-5-en-3-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), distearoylphosphatidylcholine (DSPC), Cholesterol,and1-[8′-(1,2-Dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethyleneglycol) (PEG-DMG).
 25. The composition of claim 24, wherein the LNPvehicle further comprises Linoleyl alcohol.
 26. The composition of claim25, wherein said CLinDMA, DSPC, Cholesterol, PEG-DMG, and Linoleylalcohol have a molar ratio of about 43/36/10/4/7.
 27. (canceled) 28.(canceled)
 29. (canceled)
 30. (canceled)
 31. (canceled)
 32. (canceled)33. A composition comprising a first lipid nanoparticle (LNP) vehicleand a second lipid nanoparticle (LNP) vehicle each having size betweenabout 50 nm and about 500 nm, wherein: a) each vehicle comprises acationic lipid, a neutral lipid, and a PEG-lipid; b) the first LNPvehicle further comprises one or more short interfering nucleic acid(siNA) molecules comprising a sense strand and a complementary antisensestrand, each strand having between 15 and 30 nucleotides in length,wherein the antisense strand comprises between 15 and 30 nucleotidesthat are complementary to a mammalian RNA sequence and the sense strandcomprises between 15 and 30 nucleotides of said mammalian RNA sequence;and c) the second LNP vehicle further comprises one or more emptycarrier molecules; wherein the lipids of the first LNP vehicles are thesame as or different from the second LNP vehicle.